Altered br-3 binding polypeptides

ABSTRACT

The present invention relates to novel BR3 binding antibodies having altered Fc effector function and/or having a mature core carbohydrate structure in the Fc region which lacks fiicose. The present invention also relates to the use of those BR3 binding antibodies and polypeptides in, e.g., methods of treatment, screening methods, diagnostic methods, assays and protein purification methods.

FIELD OF THE INVENTION

The invention relates to novel compositions comprising antibodies thatbind BR3, wherein the antibodies have altered Fc sequences and/orwherein the antibodies in the composition are underfucosylated, and usesthereof.

BACKGROUND OF THE INVENTION

BAFF (also known as BLyS, TALL-1, THANK, TNFSF13B, or zTNF4) is a memberof the TNF ligand superfamily that is essential for B cell survival andmaturation (reviewed in Mackay & Browning (2002) Nature Rev. Immunol. 2,465-475). BAFF overexpression in transgenic mice leads to B cellhyperplasia and development of severe autoimmune disease (Mackay, et al.(1999) J. Exp. Med. 190, 1697-1710; Gross, et al. (2000) Nature 404,995-999; Clare, et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97,3370-33752-4). BAFF levels are elevated in human patients with a varietyof autoimmune disorders, such as systemic lupus erythematosus,rheumatoid arthritis, Wegener's granulomatosis and Sjögren's syndrome(Cheema, G. S, et al., (2001) Arthritis Rheum. 44, 1313-1319; Groom, J.,et al. (2002) J. Clin. Invest. 109, 59-68; Zhang, J., et al., (2001) J.Immunol. 166, 6-10; Krumbholz et al., ANCA Workshop, Prague, CzechRepublic, 2003). Furthermore, BAFF levels correlate with diseaseseverity, suggesting that BAFF may play a direct role in thepathogenesis of these illnesses. BAFF blockade in animal models ofcollagen-induced arthritis (CIA), lupus (e.g., BWF1 mice), multiplesclerosis (e.g., experimental autoimmune encephalomyelitis (EAE))resulted in an alleviation of the disease. BR3:Fc treatment in a chronicgraft-versus-host disease (cGVHD) model significantly inhibitedsplenomegaly associated with cGVHD, not by preventing B cell activation,but by inhibiting B cell survival (Kalled, S L et al. (2005) Curr DirAutoimmun. 8:206-42). Thus, it is likely that BAFF blockade will provideefficacy in other animal models of autoimmunity with a strong B cellcomponent.

In addition, there have been reports that both CD4⁺ and CD8⁺T cells canbe costimulated by recombinant BAFF to produce Type I and Type IIcytokines and increase CD25 expression (Ng, L G, et al. 2004. J Immunol173:807). Further, BAFF-R:Fc reportedly blocked BAFF-mediated human Tcell proliferation (Huard, B, et al., (2000) J Immunol 167:6225). Stillfurther, some patients with B-lymphoid malignancies have elevated levelsof BAFF (Kern, C et al., (2004) Blood 103(2):679-88). According to onereport, adding soluble BAFF or APRIL protected B-CLL cells againstspontaneous and drug-induced apoptosis and stimulated NF-kappaBactivation. Conversely, adding soluble BCMA-Fc or anti-BAFF andanti-APRIL antibodies enhanced B-CLL apoptosis (Kern, C et al., supra).BAFF may act as an essential autocrine survival factor for malignant Bcells (Mackay F, et al., (2004) Curr Opin Pharmacol. 4(4):347-54). Thus,BAFF has been linked to a variety of disease states.

BAFF binds to three members of the TNF receptor superfamily, TACI, BCMA,and BR3 (also known as BAFF-R) (Gross, et al., supra; 8. Thompson, J.S., et al., (2001) Science 293, 2108-2111. Yan, M., et al.; (2001) Curr.Biol. 11, 1547-1552; Yan, M., et al., (2000) Nat. Immunol. 1, 37-41.Schiemann, B., et al., (2001) Science 293, 2111-2114). Of the three,only BR3 is specific for BAFF; the other two also bind the related TNFfamily member, APRIL. Comparison of the phenotypes of BAFF and receptorknockout or mutant mice indicates that signaling through BR3 mediatesthe B cell survival functions of BAFF (Thompson, et al., supra; Yan,(2002), supra; Schiemann, supra). In contrast, TACI appears to act as aninhibitory receptor (Yan, M., (2001) Nat. Immunol. 2, 638-643), whilethe role of BCMA is less clear (Schiemann, supra).

BR3 is a 184-residue type III transmembrane protein expressed on thesurface of B cells (Thompson, et al., supra; Yan, (2002), supra). Theintracellular region bears no sequence similarity to known structuraldomains or protein-protein interaction motifs. Several lines ofinvestigation have provided strong evidence that BR3 is the primaryreceptor through which B cells receive a BAFF-mediated survival signal(reviewed in Kalled, S., et al., Curr Dir Autoimmun. 2005; 8:206-42).This has been confirmed by the recent generation of BAFF-R knockout mice(Shulga-Morkskaya, S. et al., (2004) J Immunol. 15; 173(4):2331-41). BR3is expressed in a variety of disease tissue including multiple myelomaand non-Hodgkin's Lymphoma (Novak, A J (2004) Blood 104:2247-2253;Novak, A J (2004) Blood 103:689-694).

SUMMARY OF THE INVENTION

The present invention provides anti-BR3 antibodies and anti-BR3 antibodycompositions comprising novel antibody sequences (described genericallyand specifically below) having altered Fc sequences compared to awild-type sequence. The anti-BR3 antibodies of the invention can beafucosylated (i.e., where the antibody includes a mature corecarbohydrate structure which lacks fucose). A composition of anti-BR3antibodies can include anywhere from 1 to 100% afucosylated antibodies.In preferred embodiments, a composition of anti-BR3 antibodies includes2%, 4%, 6%, 10%, 19%, 20%, or anywhere up to 100% afucosylatedantibodies. In one embodiment, the percent afucosylation includes onlythe measurement of the G0-F (also known as G0-Fuc) content. In anotherembodiment, the percent afucoslyation includes the measurement of theG0-F and the G1-F or G2-F content or both. In additional preferredembodiments, the invention includes an underfucosylated composition ofanti-BR3 antibodies, where 20-100% of the N-linked oligosaccharidemolecules released from the anti-BR3 antibody composition comprise amature core carbohydrate structure which lacks fucose, attached to theFc region of the antibody. Such compositions were demonstrated herein toexhibit an improvement in binding inter alia to FcγRIIIA(F158), which isnot as effective as FcγRIIIA(V158) in interacting with human IgG.

Afucosylated antibodies or underfucosylated antibody compositions ofthis invention can be derived from a variety of methods, e.g., includingexpression of the antibodies from a cell line (e.g., CHO cells) having agene involved in the fucosyl synthesis or transfer pathway removed fromits genome (e.g., a FUT8 gene) or having such gene silenced byRNAi-mediated gene silencing or having such gene inhibited fromexpression using an inhibitor of such fucosyl synthase or transferase.Antibody compositions of this invention are useful as potent agents fortherapeutic, diagnostic or research use. The present invention furtherprovides variant BR3-binding polypeptides and compositions havingaltered Fc sequences useful for therapeutic diagnostic or research use.The present invention includes novel anti-BR3 antibody compositions,which compositions have potent ADCC activity against B cells whilelacking neutrophil killing activity compared to other B cell depletingtherapeutics, such as anti-CD20 antibodies.

According to one embodiment, the anti-BR3 binding antibodies of thisinvention comprise amino acid substitutions in their Fc region at 239and 332 (EU numbering). According to another embodiment, the anti-BR3binding polypeptides of this invention comprise amino acid substitutionsin their Fc region at 239, 298 and 332 (EU numbering). According toanother embodiment, the altered Fc sequence region comprises amino acidsubstitution(s) that increase ADCC activity relative to wild typesequence selected from the group consisting of (EU numbering): 268D,326D, 333A/334A, 298A/333A, 298A/334A, 239D/332E, 239D/298A/332E,239D/268D/298A/332E, 239D/268D/298A/326A/332A, 239D/268D/298A/326A/332E,239D/268D/283L/298A/332E, 239D/268D/283L/298A/326A/332E and239D/330L/332E of the Fc region. According to one specific embodiment,the anti-BR3 antibody further comprises altered Fc sequences thatincrease FcRn binding relative to a wild-type Fc sequence selected fromthe group consisting of N434A, N434F, N4343Y, N434W, N434H,272Y/254T/256E and T250Q/M428L. According to another embodiment, thesubstitutions are to decrease ADCC activity such as D265A (in theabsence of N297A) or N297A (in the absence of D265A). According toanother embodiment, the substitutions are S298A/E333A/K334A orS298A/K326A/E333A/K334A. According to another embodiment, thesubstitution is K322A. According one preferred embodiment, the Fcvariant increases the ADCC activity of the anti-BR3 antibody compared to9.1RF. According to another embodiment, the Fc-mutations are asdescribed elsewhere herein. According to one embodiment, the anti-BR3binding antibody has an IgG Fc sequence of human IgG1.

According to another embodiment, the anti-BR3 antibodies of thisinvention comprise variant Fc sequences that increase ADCC activity andare underfucosylated. According to one embodiment, the underfucosylatedcompositions comprise anti-BR3 antibodies with variant Fc sequences atpositions selected from the group consisting of 434, 298/326/333/334 and239/332. In another embodiment the afucosylated anti-BR3 is Hu9.1RFwhich comprises the VH and VL sequences of SEQ ID NOs: 35 and 21,respectively (see Table 2).

According to another embodiment, the anti-BR3 antibodies include anyone, any combination, or all of the following additional properties: (1)binds to a human BR3 extracellular domain sequence with an apparent Kdvalue of 500 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, 5nM or less or 1 nM or less; (2) binds to a human BR3 extracellulardomain sequence and binds to a mouse BR3 extracellular domain sequencewith an apparent Kd value of 500 nM or less, 100nM or less, 50 nM orless, 10 nM or less, 5 nM or less or 1 nM or less; (3) has a functionalepitope on human BR3 comprising a specific residue(s); (4) inhibits thebinding of human BR3 to human BAFF; (5) has increased ADCC in thepresence of human effector cells compared to wild-type IgG; (6) isderived from any one of the antibodies disclosed herein; (7) binds thehuman Fc neonatal receptor (FcRn) with a higher affinity than apolypeptide or parent polypeptide having wild type or native sequenceIgG Fc; and (8) kills or depletes B cells in vitro or in vivo,preferably by at least 20% when compared to the baseline level orappropriate negative control which is not treated with such antibody.BR3 binding polypeptides include peptides that bind BR3 (e.g., derivedfrom phage display) that are fused to Fc domains (e.g., peptibodies).

In one embodiment, compared to treatment with a control antibody thatdoes not bind a B cell surface antigen or as compared to the baselinelevel before treatment, an anti-BR3 antibody of this invention candeplete at least 20% of the B cells in any one, any combination or allof following population of cells in mice: (1) B cells in blood, (2) Bcells in the lymph nodes, (3) follicular B cells in the spleen and (4)marginal zone B cells in the spleen. In other embodiments, B celldepletion is 25%, 30%, 40%, 50%, 60%, 70%, 80% or greater.

According to one embodiment, the anti-BR3 antibodies of this inventionhave a functional epitope on human BR3 comprising residues F25, V33 andA34, wherein the monoclonal antibody is not the 9.1 antibody or the 2.1antibody. According to a further embodiment, the functional epitopefurther comprises residue R30. According to one embodiment, the BR3binding antibodies of this invention have a functional epitope on humanBR3 comprising residues P21 and A22. According to one embodiment, theBR3 binding antibodies of this invention have a functional epitope onhuman BR3 comprising residues L38 and R39, wherein the antibody is notthe 9.1 antibody. According to another embodiment, the BR3 bindingantibodies have a functional epitope on human BR3 comprising residueG36, wherein the antibody is not the 2.1 antibody. According to afurther embodiment, the BR3 binding antibodies of this invention have afunctional epitope on human BR3 comprising residues V29 and L28.According to yet another embodiment, the functional epitope furthercomprises L28 and V29. According to one embodiment, the anti-BR3antibody that has a functional epitope on human BR3 that comprises anyone, any combination or all of L38, R39, P21 and A22 is an antagonisticBR3 binding antibody.

The present invention includes anti-BR3 antibodies of Table 2, anti-BR3antibodies derived from those antibodies and antibodies that bind BR3and have an H1, H2, 1-13, L1, L2 or L3 region with at least 70% homologyto any one of the underlined portions of the antibody sequencesdescribed in the Figures or to the CDRs or hypervariable regionsdescribed in the Sequence Listing. According to one embodiment, anantibody of this invention binds BR3 and has H1, H2 and H3 regions withat least 70% homology to the H1, H2 and H3 region, respectively, of anyone of the antibodies of Table 2. According to one embodiment, anantibody of this invention binds BR3 and has L1, L2 and L3 regions withat least 70% homology to the L1, L2 and L3 region, respectively, of anyone of the antibodies of Table 2. According to another embodiment, theantibodies bind BR3 and have a VH domain with at least 70% homology to aVH domain of any one of the antibodies of Table 2.

The present invention provides humanized anti-BR3 antibodies comprisingan H3 hypervariable region (HVR3) comprising the residues QVRRALDY (SEQID NO:212). According to another embodiment, an anti-BR3 antibodycomprises: (1) an H3 hypervariable region (HVR3) comprising the residuesQVRRALDY (SEQ ID NO:212); and (2) a heavy chain framework 3 region(HC-FR3) comprising the residues RDTSKNTF (SEQ ID NO:210). In oneembodiment, the BR3 binding antibody further comprises an HVR1comprising residues numbered 26-35 and an HVR2 comprising residues 49-65(Kabat numbering) of an antibody sequence of any one of SEQ ID NOs:35-36. In another embodiment, the anti-BR3 antibody further comprisesresidues GFTVTAYYMS (SEQ ID NO:214) in the H1 hypervariable region(HVR1) and residues GFIRDKANGYTTEYNPSVKG (SEQ ID NO: 213) in the H2hypervariable region (HVR2). According to one embodiment, the antibodyfurther comprises residues KSSQSLLYSSNQNNYLA (SEQ ID NO:232) in theLVR1, residues WASTRES (SEQ ID NO:233) in the LVR2 and residuesQQSQISPPT (SEQ ID NO:231) in the LVR3.

According to another embodiment, an anti-BR3 binding antibody of theinvention comprises: (1) an H3 hypervariable region (HVR3) comprisingQVRRALDY (SEQ ID NO:212); and (2) a heavy chain framework 3 region(HC-FR3) comprising RDTSKNTL (SEQ ID NO:211). In one embodiment, the BR3binding antibody comprises residues numbered 26-35 and 49-65 (Kabatnumbering) of any one of the antibody sequences of SEQ ID NOs:37-73.According to one embodiment, the antibody further comprises residuesKSSQSLLYSSNQNNYLA (SEQ ID NO:232) in the LVR1, residues WASTRES (SEQ IDNO:233) in the LVR2 and residues QQSQISPPT (SEQ ID NO:231) in the LVR3.

According to another embodiment, an anti-BR3 binding antibody of theinvention comprises an L2 hypervariable region (LVR2) comprising FormulaI:

W-A-X3-X3-X4-X3X5-X6-S (SEQ ID NO:215)  (Formula I),

wherein X3 is Q or S; X4 is H, I or T; X5 is L or R and X6 is D or E andwherein Formula I is not WASTRES (SEQ ID NO:233). According to oneembodiment, the anti-BR3 antibody further comprises an H3 hypervariableregion (HVR3) comprising QVRRALDY (SEQ ID NO:212). According to oneembodiment, the LVR2 comprises residues numbered 50-56 (Kabat numbering)of the antibody sequence selected from the group consisting of SEQ IDNOs:23 and 25. According to another embodiment, the antibody furthercomprises residues GFTVTAYYMS (SEQ ID NO:214) in the HVR1 and residuesGFIRDKANGYTTEYNPSVKG (SEQ ID NO:213) in the HVR2. According to yetanother embodiment, the antibody further comprises residuesKSSQSLLYSSNQNNYLA (SEQ ID NO:232) in the LVR1 and residues QQSQISPPT(SEQ ID NO:231) in the LVR3.

According to another embodiment, an anti-BR3 binding antibody of theinvention comprises: an H1 hypervariable region (HVR1) comprisingFormula II:

X1-X2-X3-X4-X5-X6-X7-Y-X9-X10 (SEQ ID NO:216)  (Formula II),

wherein X1 is G or D, S, A, V, E or T; X2 is L, S, W, P, F, A, V, I, R,Y or D; X3 is P, T, A, N, S, I, K, L or Q; X4 is M, R, V, Y, G, E, A, T,L, W or D; X5 is A, S, T, G, I, R, P, N, D, Y or H; X6 is G, A, S, P orT; X7 is F, H, Y, R, S, V or N; X9 is T, I, M, F, W or V; X10 is T, G, Sor A and wherein Formula II is not GFTVTAYYMS (SEQ ID NO:214). Accordingto one embodiment, the antibody further comprises an H3 hypervariableregion (HVR3) comprising QVRRALDY (SEQ ID NO:212). According to oneembodiment, the HVR1 comprises residues numbered 26-35 (Kabat numbering)of the antibody sequence selected from the group consisting of SEQ IDNOs:24, 26-34, 36 and 38-73. According to one embodiment, the antibodyfurther comprises residues WASTRES (SEQ ID NO:233) in the LVR2.According to one embodiment, the antibody further comprises residuesKSSQSLLYSSNQNNYLA (SEQ ID NO:232) in the LVR1, residues WASTRES (SEQ IDNO:233) in the LVR2 and residues QQSQISPPT (SEQ ID NO:231) in the LVR3.According to one embodiment, the antibody further comprises residuesGFIRDKANGYTTEYNPSVKG (SEQ ID NO:213) in the HVR2.

According to another embodiment, an anti-BR3 binding antibody of thisinvention is an antibody that comprises: (1) an H3 hypervariable region(HVR3) comprising QVRRALDY (SEQ ID, NO:212) and (2) residues numbered50-56 of the LVR2 and residues numbered 26-35 of the HVR1 of an antibodyselected from the group consisting of Hu9.1-73, Hu9.1-70, Hu9.1-56,Hu9.1-51, Hu9.1-59, Hu9.1-61, Hu9.1-A, Hu9.1-B and Hu9.1-C. According toone embodiment, the antibody further comprises residuesGFIRDKANGYTTEYNPSVKG (SEQ ID NO:213) in the HVR2. According to oneembodiment, the antibody further comprises residues KSSQSLLYSSNQNNYLA(SEQ ID NO:232) in the LVR1 and residues QQSQISPPT (SEQ ID NO:231) inthe LVR3.

The present invention also provides anti-BR3 antibodies comprising anHVR3 comprising residues numbered 94-102 (Kabat numbering) of theantibody sequence selected from the group consisting of SEQ ID NOS:7-13and 16-18. According to one embodiment, the antibody further comprisesan HVR1 and HVR2 comprising residues 26-35 and residues 49-65 (Kabatnumbering), respectively, of the antibody sequence of any one of SEQ IDNOS:7-13 and 16-18. According to one embodiment, the LVR1, LVR2 and LVR3of the antibody comprises residues 24-34, residues 50-56 and residues89-97 (Kabat numbering), respectively, of the antibody sequence of SEQID NO:3.

According to one embodiment, the anti-BR3 comprises a variable heavychain domain comprising the variable heavy chain sequence of any one ofSEQ ID NOs:22, 24 and 26-73. According to one embodiment, the anti-BR3comprises a variable light chain domain comprising the variable lightchain sequence of any one of SEQ ID NOs:21, 23 and 25. According toanother embodiment, the antibody comprises the sequence of SEQ ID NO:74.According to another embodiment, the antibody comprises the sequence ofSEQ ID NO:76, wherein X is N, A, W, H, Y, S or F. According to onespecific embodiment, the antibody comprises the sequence of SEQ IDNO:75.

The present invention also provides an anti-BR3 antibody comprising anHVR3 comprising Formula III:

X1-X2-X3-X4-X5-G-X7-MDY (SEQ ID NO:218)  (Formula III),

wherein X1 is N, T or R; X2 is A, S, T, L, N or P; X3 is N, H or L; X4is P, Y, F, N, T or L; X5 is Y, T or D; and X7 is A or E. According toone embodiment, Formula III is not TPHTYGAMDY (SEQ ID NO:235). Accordingto one embodiment, Formula III is NSNFYGAMDY (SEQ ID NO:219). Accordingto one embodiment, the antibody further comprises an HC-FR3 comprisingresidues RDTSKNTF (SEQ ID NO:210) or RDTSKNTL (SEQ ID NO:211). Accordingto one embodiment, the LVR1, LVR2 and LVR3 of the antibody compriseresidues 24-34, residues 50-56 and residues 89-97 (Kabat numbering),respectively, of the antibody sequence of SEQ ID NO:3. According to oneembodiment, the HVR1 and HVR2 of the antibody comprise residues 26-35and residues 49-65 (Kabat numbering), respectively, of the antibodysequence of SEQ ID NO:4.

Alternatively, the present invention provides an anti-BR3 antibodycomprising an HVR3 comprising Formula III:

X1-X2-X3-X4-X5-G-X7-MDY (SEQ ID NO:218)  (Formula III),

wherein X1 is N, T or R; X2 is A, S, T, L, N or P; X3 is N, 1-1 or L; X4is P, Y, F, N, T or L; X5 is Y, T or D; and X7 is A or E and wherein theantibody further comprises an HC-FR3 comprising residues RDTSKNTF (SEQID NO:210) or RDTSKNTL (SEQ ID NO:211). According to one embodiment,when the HC-FR3 comprises RDTSKNTF (SEQ ID NO:210), then HVR3 of theantibody comprises residues 94-102 (Kabat numbering) of the antibodysequence of any one of SEQ ID NOs:6-9 and 16-17. According to oneembodiment, when the HC-FR3 comprises RDTSKNTL (SEQ ID NO:211), then theHVR3 of the antibody comprises residues 94-102 (Kabat numbering) of theantibody sequence of any one of SEQ ID NOs:5 and 10-13. According to oneembodiment, the LVR1, LVR2 and LVR3 of the antibody comprise residues24-34, residues 50-56 and residues 89-97 (Kabat numbering),respectively, of the antibody sequence of SEQ ID NO:3. According to oneembodiment, the HVR1 and HVR2 of the antibody comprise residues 26-35and residues 49-65 (Kabat numbering) of the antibody sequence of SEQ IDNO:4, respectively.

In one embodiment, the sequence of Formula III is Formula IV:

X1-X2-X3-X4-X5-GAMDY (SEQ ID NO:217)  (Formula IV),

wherein X1 is N, T or R; X2 is S, T, L, N or P; X3 is N or L; X4 is P,Y, F, N or L; X5 is Y or D.

According to one embodiment, the anti-BR3 antibody of the inventioncomprises an HVR3 comprising the sequence of Formula IV and an HC-FR3comprising the sequence of SEQ ID NO:210. In a further embodiment, theantibody comprises the light chain sequence of SEQ ID NO:14. In afurther embodiment, the antibody comprises an Fc region havingD265A/N297A (EU numbering) mutations or another Fc mutation thatdecreases ADCC activity of the antibody.

According to one embodiment, the anti-BR3 antibody of the inventioncomprises a variable heavy chain domain comprising the variable heavychain sequence of any one of SEQ ID NOs:4-13 and 16-18. According to oneembodiment, the anti-BR3 comprises a variable light chain domaincomprising the variable light chain sequence of SEQ ID NO:3. Accordingto another embodiment, the antibody comprises the sequence of SEQ IDNO:14. According to another embodiment, the antibody comprises thesequence of SEQ ID NO:15.

The present invention further provides an anti-BR3 antibody of theinvention comprises the variable light chain sequence SEQ ID NO:77 andthe variable heavy chain sequence SEQ ID NO:78, and variants thereof.According to one embodiment, an anti-BR3 antibody comprises the variablelight chain sequence of SEQ ID NO:79. According to another embodiment,an anti-BR3 antibody comprises the variable heavy chain sequence of anyone of SEQ ID NOs:80-85. According to one embodiment, an anti-BR3antibody comprises an HVR1 comprising residues numbered 26-35 (Kabatnumbering) of the antibody sequence of any one of SEQ ID NOs:80 or 82.According to one embodiment, an anti-BR3 antibody comprises an HVR2comprising residues numbered 49-65 (Kabat numbering) of the antibodysequence of any one of SEQ ID NOs:80, 84 or 85. According to oneembodiment, an anti-BR3 antibody comprises an HVR3 comprising residuesnumbered 94-102 (Kabat numbering) of the antibody sequence of any one ofSEQ ID NOs:80, 82 or 83. In another embodiment, the anti-BR3 antibodycomprises (1) an HVR3 comprising residues 94-102 (Kabat numbering) ofthe antibody sequence of any one of SEQ ID NOs: 81-85 and (2) a heavychain framework 3 region (HC-FR3) comprising RDTSKNTF (SEQ ID NO:210).According to one embodiment, an anti-BR3 antibody comprises residuesnumbered 26-35, 49-65 and 94-102 of the antibody sequence of any one ofSEQ ID NOs:80-85. According to one embodiment, the anti-BR3 antibodycomprises an LVR1 comprising residues numbered 24-34 (Kabat numbering)of the antibody sequence SEQ ID NO:79. According to one embodiment, theanti-BR3 antibody comprises an LVR2 comprising residues numbered 50-56(Kabat numbering) of the antibody sequence SEQ ID NO:79. According toone embodiment, the anti-BR3 antibody comprises an LVR3 comprisingresidues numbered 89-97 (Kabat numbering) of the antibody sequence SEQID NO:79. According to another embodiment, the LVR1, LVR2 and LVR3 of ananti-BR3 antibody comprises residues numbered 24-34, 50-56 and 89-97(Kabat numbering), respectively, of SEQ ID NO:79.

According to one embodiment, the anti-BR3 comprises a variable heavychain domain comprising the variable heavy chain sequence of any one ofSEQ ID NOs:78 and 80-85. According to one embodiment, the anti-BR3antibody comprises a variable light chain domain comprising the variablelight chain sequence of SEQ ID NO:77 and 79.

The present invention also provides an anti-BR3 antibody comprising anHVR3 comprising residues numbered 95-102 of the antibody sequence of anyone of SEQ ID NOs:87-94. The present invention provides an anti-BR3antibody comprising an HVR2 comprising residues numbered 49-58 of theantibody sequence of any one of SEQ ID NOs:87-94, 98, 100, 102, 104,106, 107, 109-110, 112, 114, 116, 118, 120, 122, 124-127, 129 and 193.The present invention provides an anti-BR3 antibody comprising an HVR1comprising residues numbered 24-34 of the antibody sequence of any oneof SEQ ID NOs:87-94, 98, 100, 102, 104, 106, 107, 109-110, 112, 114,116, 118, 120, 122, 124-127, 129 and 193. The present invention providesan anti-BR3 antibody comprising an LVR1 comprising residues numbered24-34 of the antibody sequence of any one of SEQ ID NOs:86, 97, 99, 101,103, 105, 108, 111, 113, 115, 117, 119, 121, 123, 128 and 194-207. Thepresent invention provides an anti-BR3 antibody comprising an LVR2comprising residues numbered 50-56 of the antibody sequence of any oneof SEQ ID NOs:86, 97, 99, 101, 103, 105, 108, 111, 113, 115, 117, 119,121, 123, 128 and 194-207. The present invention provides an anti-BR3antibody comprising an LVR3 comprising residues numbered 89-97 of theantibody sequence of any one of SEQ ID NOs:86, 97, 99, 101, 103, 105,108, 111, 113, 115, 117, 119, 121, 123, 128 and 194-207. According toone embodiment, the LVR1, LVR2 and LVR3 comprise residues numbered24-34, 50-56 and 89-97 of the antibody sequence of any one of SEQ IDNOs:86, 97, 99, 101, 103, 105, 108, 111, 113, 115, 117, 119, 121, 123,128 and 194-207. According to one embodiment, the HVR1, HVR2 and HVR3comprise residues numbered 24-34, 49-58 and 95-102 of the antibodysequence of any one of SEQ ID NOs:87-94, 98, 100, 102, 104, 106, 107,109-110, 112, 114, 116, 118, 120, 122, 124-127, 129 and 193. In oneembodiment, the anti-BR3 antibody comprises a variable heavy chaindomain comprising the VH sequence of any one of SEQ ID NOs:87-94, 98,100, 102, 104, 106, 107, 109-110, 112, 114, 116, 118, 120, 122, 124-127,129 and 193. In another embodiment, the anti-BR3 antibody comprises avariable light chain domain comprising the VL sequence of any one of SEQID NOs:86, 97, 99, 101, 103, 105, 108, 111, 113, 115, 117, 119, 121,123, 128 and 194-207.

The present invention further provides an anti-BR3 antibody comprisingHVR3 comprising RVCYN-X6-LGVCAGGMDY (SEQ ID NO:220) (Formula V), whereinX6 is R or H.

The present invention provides an anti-BR3 antibody comprising an LVR1comprising the Formula VI:

RAS-X4-X5-X6-X7-X8-X9-VA  (Formula VI) (SEQ ID NO: 226),

wherein X4 is Q or E; X5 is D or E; X6 is I or E; X7 is S or A, X8 is Sor T and X9 is A or S.

The present invention provides an anti-BR3 antibody comprising an LVR2comprising the Formula VII:

X1-X2-A-S—X5-L-X7-S  (Formula VII) (SEQ ID NO: 227),

Wherein X1 is Y or F; X2 is S, A or G; X5 is N, F or Y; and X7 is F orY.

The present invention provides an anti-BR3 antibody comprising an LVR3comprising the Formula VIII:

Q-X2-S—X4-X5-X6-PPT  (Formula VIII) (SEQ ID NO: 228),

wherein X2 is Q or H; X4 is G, L, R, H, Y, Q or E; X5 is N, T, M, S, A,T, I or V; and X6 is T or S. According to one embodiment, the anti-BR3antibody comprises a light chain comprising the sequences of Formula I,II and III. According to another embodiment, the anti-BR3 antibodycomprises a light chain comprising the sequences of Formula I, II andIII and comprises a HVR3 comprising the sequence of Formula V or SEQ IDNO:220.

The present invention also provides an anti-BR3 binding antibody thatcomprises an H3 comprising RVCYNRLGVCAGGMDY (SEQ ID NO:221); an H1comprising residues SGFTISSNSIH (SEQ ID NO:222) and an H2 comprisingAWITPSDGNTD (SEQ ID NO: 223). In another embodiment, the anti-BR3binding antibody comprises an H3 comprising RVCYNRLGVCAGGMDY (SEQ IDNO:221); an H1 comprising residues SGFTISSSSIH (SEQ ID NO:224) and an H2comprising AWVLPSVGFTD (SEQ ID NO: 225).

According to one embodiment, the anti-BR3 comprises a variable heavychain comprising the variable heavy chain sequence of any one of SEQ IDNOs:87-96, 98, 100, 102, 104, 106, 107, 109-110, 112, 114, 116, 118,120, 122, 124-127, 129 and 193. According to one embodiment, theanti-BR3 antibody comprises a variable light chain comprising thevariable light chain sequence of any one of SEQ ID NOs:86, 97, 99, 101,103, 105, 108, 111, 113, 115, 117, 119, 121, 123, 128 and 194-207.

In one embodiment, the anti-BR3 antibody can competitively inhibit thebinding of an antibody produced by the hybridoma deposited as 3.1 (ATCCDeposit PTA-6622) or 12B12.1 (ATCC Deposit PTA-6624) to the human BR3extracellular domain. In a further embodiment, the antibody comprisesthe variable region sequence of the antibody produced by the hybridomadeposited as 3.1 (ATCC Deposit PTA-6622) or 12B12.1 (ATCC DepositPTA-6624) to the human BR3 extracellular domain. In another embodiment,the antibody comprises the hypervariable region sequence of the antibodyproduced by the hybridoma deposited as 3.1 (ATCC Deposit PTA-6622) or12B12.1 (ATCC Deposit PTA-6624). In another embodiment, the antibody isa humanized form of the antibody produced by the hybridoma deposited as3.1 (ATCC Deposit PTA-6622) or 12B12.1 (ATCC Deposit PTA-6624).

In one embodiment, the anti-BR3 antibody can competitively inhibit thebinding of an antibody produced by the hybridoma deposited as 3.1 (ATCCDeposit PTA-6622) or 12B12.1 (ATCC Deposit PTA-6624) to human BR3. In afurther embodiment, the antibody comprises the variable region sequenceof the antibody produced by the hybridoma deposited as 3.1 (ATCC DepositPTA-6622) or 12B12.1 (ATCC Deposit PTA-6624) to human BR3. In anotherembodiment, the antibody comprises the hypervariable region sequence ofthe antibody produced by the hybridoma deposited as 3.1 (ATCC DepositPTA-6622) or 12B12.1 (ATCC Deposit PTA-6624). In another embodiment,antibody is a humanized form of the antibody produced by the hybridomadeposited as 3.1 (ATCC Deposit PTA-6622) or 12B12.1 (ATCC DepositPTA-6624).

In one embodiment, the antibody of this invention binds to the sameepitope as any one of the antibodies specifically described herein. Inanother embodiment, the antibody of this invention comprises thesequence of the deposited antibodies.

According to one embodiment of the invention, the anti-BR3 antibody isconjugated to a cytotoxic agent or a chemotherapeutic agent.

According to another embodiment, the antibody is a monoclonal antibody.According to another embodiment, the antibody is a humanized antibody.According to another embodiment, the antibody is a human antibody.According to another embodiment, the antibody is a chimeric antibody.According to another embodiment, the antibody is selected from the groupconsisting of a Fab, Fab′, a F(ab)′₂, single-chain Fv (scFv), an Fvfragment; a antiabody and a linear antibody. According to anotherembodiment, the antibody is a multi-specific antibody such as abispecific antibody.

Also provided is a composition comprising an antibody of any one of thepreceding embodiments, and a carrier. In one embodiment, the carrier isa pharmaceutically acceptable carrier. These compositions can beprovided in an article of manufacture or a kit.

The invention also provided a liquid formulation comprising an anti-BR3antibody in a histidine buffer. According to one embodiment, the bufferis a histidine sulfate buffer. According to another embodiment, aformulation or composition of this invention is packaged as a pre-filledsyringe.

The invention also provides an isolated nucleic acid that encodes any ofthe antibody sequences disclosed herein, including an expression vectorfor expressing the antibody.

Another aspect of the invention are host cells comprising the precedingnucleic acids, and host cells that produce the antibody. In onepreferred embodiment of the latter, the host cell is a CHO cell. Amethod of producing these antibodies is provided, the method comprisingculturing the host cell that produces the antibody and recovering theantibody from the cell culture.

Yet another aspect of the invention is an article of manufacturecomprising a container and a composition contained therein and a packageinsert, wherein the composition comprises an antibody of any of thepreceding embodiments. According to one embodiment, the article ofmanufacture is a diagnostic kit comprising a BR3-binding antibody ofthis invention.

The invention also provides methods of treating the diseases disclosedherein by administration of an anti-BR3 antibody of the invention,polypeptide or functional fragment thereof, to a mammal such as a humanpatient having a bone marrow transplant and a human patient sufferingfrom the disease such as an autoimmune disease, a cancer, a B cellneoplasm, a BR3 positive cancer or an immunodeficiency disease.According to one preferred embodiment for treating an autoimmunedisease, B cell neoplasm or a BR3 positive cancer, the BR3 bindingpolypeptide or antibody to be administered is preferably an antagonistBR3-binding antibody or polypeptide or is not an agonist BR3 bindingantibody or polypeptide. According to one embodiment, the cancer to betreated according to this invention is selected from the groupconsisting of non-Hodgkin's lymphoma, chronic lymphocytic leukemia,multiple myeloma, (including follicular lymphoma, diffuse large B celllymphoma, marginal zone lymphoma and mantle cell lymphoma).

In one embodiment of the methods for treating an autoimmune disease,cancer, B cell neoplasm or a BR3 positive cancer, the antibody is aBR3-binding antibody that has increased ability to bind FcRn at pH 6.0compared to a 9.1RF antibody of this invention. In one embodiment of themethods for treating an autoimmune disease, B cell neoplasm or a BR3positive cancer, the BR3 binding antibody is a BR3-binding antibody thathas increased ADCC effector function in the presence of human effectorcells compared to a 9.1RF antibody.

In one embodiment, the BR3 positive cancer is a B cell lymphoma orleukemia including non-Hodgkin's lymphoma (NHL) or lymphocytepredominant Hodgkin's disease (LPHD), chronic lymphocytic leukemia(CLL), acute lymphocytic leukemia (ALL) or small lymphocytic lymphoma(SLL). According to another embodiment, the BR3 positive cancer ismultiple myeloma. In additional embodiments, the treatment methodfurther comprises administering to the patient at least onechemotherapeutic agent, wherein for non-Hodgkin's lymphoma (NHL), thechemotherapeutic agent is selected from the group consisting ofdoxorubicin, cyclophosphamide, vincristine and prednisolone.

Also provided is a method of treating an autoimmune disease, comprisingadministering to a patient suffering from the autoimmune disease, atherapeutically effective amount of a BR3 binding antibody orpolypeptide of this invention. According to one embodiment, theautoimmune disease is selected from the group consisting of rheumatoidarthritis, juvenile rheumatoid arthritis, lupus including systemic lupuserythematosus (SLE), Wegener's disease, inflammatory bowel diseaseincluding Crohn's Disease and ulcerative colitis, idiopathicthrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura(TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, Igneuropathies including IgA nephropathy, IgM polyneuropathies, and IgGneuropathy, myasthenia gravis, vasculitis including ANCA-associatedvasculitis, diabetes mellitus, Reynaud's syndrome, Sjorgen's syndrome,neuromyelitis optica (NMO), pemphigus including paraneoplasticpemphigus, pemphigus vulgaris and pemphigus foliaceus,polymyositis/dermatomyositis and glomerulonephritis. Where theautoimmune disease is rheumatoid arthritis, the antibody can beadministered in conjunction with a second therapeutic agent. Accordingto one embodiment, the second therapeutic agent is methotrexate.

In these treatment methods for autoimmune diseases, B cell neoplasms,BR3 positive cancers, the BR3 binding antibodies can be administeredalone or in conjunction with a second therapeutic agent such as a secondantibody, another B cell depleting agent, a chemotherapeutic agent, animmunosuppressive agent or another biologic that modulates human immuneresponses (e.g., a biologic response modifier). The second antibody canbe one that binds CD20 or a different B cell antigen, or an NK or T cellantigen. In one embodiment, the anti-CD20 antibody is selected from thegroup consisting of rituximab (Rituxan®), m2H7 (murine 2H7), hu2H7(humanized 2H7) and all its functional variants, hu2H7.v16 (v stands forversion), v31, v96, v114 and v115, (e.g., see, WO 2004/056312). In oneembodiment, the second antibody is a radiolabeled anti-CD20 antibody. Inother embodiments, the CD20 binding antibody is conjugated to acytotoxic agent including a toxin or a radioactive isotope. In anotherembodiment, the second therapeutic agent is selected from the groupconsisting of an interleukin (e.g., IL-2, IL-12), an interferon,fludarabine, cyclophosphamide, an antibody that targets TNF-alpha (e.g.,Enbrel®, Remicade®, and Humira®), or a colony-stimulating factor (e.g.,CSF, GM-CSF, G-CSF). In another embodiment, the second antibody orbiologic can be another BAFF antagonist (e.g., a BR3 antibody, anti-BAFFantibody, TACI-Fc, BCMA-Fc and BR3-Fc). According to one embodiment, theBAFF antagonist that is being administered as a second therapeutic forautoimmune diseases or cancer does not have ADCC activity. In anotherembodiment, the second therapeutic is selected from the group consistingof an anti-VEGF antibody (e.g., the Avastin™ antibody), anti-CD64antibody, an anti-C32a antibody, an anti-CD16 antibody, anti-INFalphaantibody, anti-CD79a antibody, an anti-CD70b antibody, an anti-CD52antibody, anti-CD40 antibody, CTLA4-Ig, anti-CD22 antibody, anti-CD23antibody, anti-CD80 antibody, anti-HLA-DR antibody, anti-MHCII (IA)antibody, anti-IL-7 antibody, anti-IL-2 antibody, anti-IL-4 antibody, ananti-IL-21 antibody and anti-IL-10 antibody. Specific examples of B celldepletion agents include, but are not limited to, the aforementionedanti-CD20 antibodies, Alemtuzumab (anti-CD52 antibody), and Epratuzumabor CMC-544 (Wyeth) (anti-CD22 antibodies). In another embodiment, thesecond therapeutic is a small molecule that depletes B cells or an IAPinhibitor.

In another aspect, the invention provides a method of treating anautoimmune disease selected from the group consisting ofDermatomyositis, Wegner's granulomatosis, ANCA-associated vasculitis(AAV), Aplastic anemia, Autoimmune hemolytic anemia (AIHA), factor VIIIdeficiency, hemophilia A, Autoimmune neutropenia, Castleman's syndrome,Goodpasture's syndrome, solid organ transplant rejection, graft versushost disease (GVHD), IgM mediated, thrombotic thrombocytopenic purpura(TTP), Hashimoto's Thyroiditis, autoimmune hepatitis, lymphoidinterstitial pneumonitis (LIP), bronchiolitis obliterans(non-transplant) vs. NSIP, Guillain-Barre Syndrome, large vesselvasculitis, giant cell (Takayasu's) arteritis, medium vessel vasculitis,Kawasaki's Disease, polyarteritis nodosa, comprising administering to apatient suffering from the disease, a therapeutically effective amountof a BR3 binding antibody.

The present invention also provides a method for diagnosing anautoimmune disease or a cancer to be treated with a BR3 binding therapyantagonist which comprises: (a) contacting a biological sample from atest subject with an anti-BR3 antibody of this invention; (b) assayingthe level of BR3 polypeptide in the biological sample; and (c) comparingthe level of BR3 polypeptide in the biological sample in the biologicalsample with a standard level of BR3 protein; whereby the presence or anincrease in the level of BR3 protein compared to the standard level ofBR3 protein is indicative of an autoimmune disease or cancer to betreated with a BR3 binding therapy.

The present invention also provides a method of detecting BR3polypeptide comprising the steps of binding the anti-BR3 antibody orimmunoadhesin of this invention in a test sample or a subject andcomparing the antibody or immunoadhesin bound compared to a controlantibody or immunoadhesin. In one embodiment, the antibody orimmunoadhesin is used in an assay selected from the group consisting ofa FACS analysis, an immunohistochemistry assay (IHC) and an ELISA assay.Non-BAFF blocking anti-BR3 antibodies have the advantage of detectingBR3 whether it is bound to ligand or not and can be useful in measuringfree and bound BR3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically depicts the results of ADCC assays with 9.1RF Fcvariants and BJAB cells.

FIGS. 2A and 2B graphically depict the results of ADCC assays with 9.1RF Fc variants and (A) BJAB or (B) WIL2-S cells.

FIG. 3 graphically depicts fold change in binding of V3-46s Fc variantsto mouse or human FcγR relative to a control.

FIGS. 4A and 4B graphically depict the ADCC activity of V3-46s Fcvariants on BJAB cells.

FIG. 5 is a schematic describing the experimental design of B celldepletion assays in mice using V3-46s Fc variants.

FIG. 6 graphically depicts the results of blood B cell depletion aftertreatment with V3-46s Fc variants.

FIG. 7 shows examples of oligosaccharide structures present on IgG,their numerical assignments in m/z via MALDI-TOF analyses.

FIG. 8 is a schematic of the configuration of the RNAi plasmids.Abbreviations: CMV, cytomegalovirus promoter and enhancer sequence; PD,PUR-DHFR; HC, heavy chain; LC, light chain.

FIG. 9 shows the RNAi probes developed to decrease fucosyltransferase(FUT8) in cells.

FIG. 10 graphically depicts the percentage of non-fucosylated antibodyin samples of 9.1RF or 9.1(5) Fc variant produced from cells transientlytransfected with or without RNAi2 and RNAi4.

FIG. 11 graphically depicts the ADCC activity of 9.1RF Fc variantsexpressed from cells transiently transfected with RNAi2 and RNAi4 ornot.

FIGS. 12A and 12B graphically depict the fold increased binding of 9.1RF Fc variants to (12A) human or (12B) cyno FcγRIIIa relative to acontrol antibody (the Herceptin® antibody).

FIGS. 13A and 13B graphically depict the direct and competitive bindingof control and afucosylated anti-BR3 mAbs to BJAB cells. FIG. 13Agraphically depicts the direct binding assay where the indicatedconcentrations of mAbs were added to BJAB cells and bound mAb wasdetected with anti-human IgG Fc-HRP. FIG. 13B graphically depicts thecompetitive binding assay where the indicated concentrations of mAbswere added to cells in the presence of 25 nM FLAG-BAFF followed bydetection of bound BAFF using anti-FLAG-HRP.

FIGS. 14A and 14B graphically depict the evaluation of the antagonisticeffects of anti-BR3 (Hu9.1 RF) and control antibodies on primary human Bcell proliferation. B cells were incubated with a dilution curve ofanti-BR3 mAbs in the presence of anti-IgM (4 μg/ml) and BAFF (20 ng/ml).In FIG. 14A, the control and afucosylated anti-BR3 antibodies caused anidentical dose-dependent inhibition of B cell proliferation. In FIG.14B, neither the agonistic positive control (“Control”-Hu9.1RF with anN434W mutation) nor the isotype negative control (Herceptin®) antibodiesinhibited B cell proliferation.

FIGS. 15A and 15B graphically depict the evaluation of potentialagonistic effects of anti-BR3 and control antibodies. B cells wereincubated with a dilution curve of antibodies in the presence ofanti-IgM (4 μg/ml) alone. In FIG. 15A, neither the control nor theafucosylated (AF) Hu9.1RF IgG1 antibody stimulated B cell proliferationabove the level elicited by treatment with anti-IgM. In FIG. 15B, theIgG1 isotype control had no effect, whereas Hu9.1RF IgG1 with an N434Wmutation (“Control”) caused a dose-dependent increase in B cellproliferation.

FIG. 16 graphically depicts the results of an ELISA showing the bindingof antibodies to human FcRN at pH 6.0. Various concentrations of controland test material were incubated with human FcRn immobilized on theassay plate at pH 6.0. All data points were collected in duplicate, andthe mean absorbance values were plotted versus the antibodyconcentration

FIG. 17 graphically depicts the results of an ELISA shown thedissociation of bound IgG at pH 6.0 or 7.4. Various concentrations ofcontrol and test material were incubated with human FcRn immobilized onthe assay plate at pH 6.0. The dissociation of bound IgG from FcRn wasevaluated after incubation in pH 6.0 or 7.4 assay buffer. All datapoints were collected in singlet, and the absorbance values were plottedversus the antibody concentration.

FIGS. 18A and 18B graphically depict the CDC activity of AF HU9.1RF IgG1negative control and positive control monoclonal antibodies on BJAB andWIL2 cells.

FIGS. 19A-19C graphically depict the ADCC activity of AF HU9.1RF IgG1with 2% afucosylated HU9.1RF IgG1 using normal human NK cells from threenormal donors.

FIGS. 20A-20C graphically depict the ADCC activity of HU9.1RF IgG1monoclonal antibodies differing in percent afucosylation using normalhuman NK cells from three normal donors.

FIGS. 21A-21C graphically depict the induction of apoptosis by AFHu9.1RF IgG1 in BR3 positive BJAB cells. AF Hu9.1RF IgG1 atconcentrations ranging from 0.1 to 100 nM in the presence of anti IgGcrosslinker did not affect the viability of the cells (FIG. 21A), thelevel of annexin staining (FIG. 21B), or the level of propidium iodidestaining (FIG. 21C) as compared to untreated cells.

DETAILED DESCRIPTION OF THE INVENTION

The carbohydrate moieties of the present invention will be describedwith reference to commonly used nomenclature for the description ofoligosaccharides. A review of carbohydrate chemistry which uses thisnomenclature is found in Hubbard et al. Ann. Rev. Biochem. 50:555-583(1981). This nomenclature includes, for instance, Man, which representsmannose; GlcNAc, which represents 2-N-acetylglucosamine; Gal whichrepresents galactose; Fuc for fucose; and Glc, which represents glucose.Sialic acids are described by the shorthand notation NeuNAc, for5-N-acetylneuraminic acid, and NeuNGc for 5-glycolylneuraminic.

The term “glycosylation” means the attachment of oligosaccharides(carbohydrates containing two or more simple sugars linked together e.g.from two to about twelve simple sugars linked together) to a protein toform a glycoprotein. The oligosaccharide side chains are typicallylinked to the backbone of the glycoprotein through either N- orO-linkages. The oligosaccharides of the present invention occurgenerally are attached to a CH2 domain of an Fc region as N-linkedoligosaccharides.

“N-linked glycosylation” refers to the attachment of the carbohydratemoiety to an asparagine residue in a glycoprotein chain. The skilledartisan will recognize that, for example, each of murine IgG1, IgG2a,IgG2b and IgG3 as well as human IgG1, IgG2, IgG3, IgG4, IgA and IgD CH2domains have a single site for N-linked glycosylation at amino acidresidue 297 (Kabat et al. Sequences of Proteins of ImmunologicalInterest, 1991).

“Glycoproteins” are polypeptides having one or more oligosaccharide sidechains attached thereto.

For the purposes herein, a “mature core carbohydrate structure” refersto a processed core carbohydrate structure attached to an Fc regionwhich generally comprises the following carbohydrate structureGlcNAc(Fucose)-GlcNAc-Man-(Man-GleNAc)₂ typical of biantennaryoligosaccharides represented schematically below:

However, this term specifically includes G-1 forms of the core maturecarbohydrate structure lacking a β1,2 GlcNAc residue. Preferably,however, the core carbohydrate structure includes both β1,2 GlcNAcresidues. The mature core carbohydrate structure herein generally is nothypermannosylated.

The mature core carbohydrate structure is attached to the Fc region ofthe glycoprotein, generally via N-linkage to Asn297 of a CH2 domain ofthe Fc region.

The term “underfucosylated” means that 20-100% of the N-linkedoligosaccharide molecules released from the Fc region of the anti-BR3antibody composition comprise a mature core carbohydrate structure whichlacks fucose (“non-fucosylated”), attached to the Fc region of theantibody. Thus, it should be understood that the term underfucosylatedincludes anti-BR3 antibody compositions having N-linked oligosaccharideswith no fucose entirely. According to one preferred embodiment, thepercentage of non-fucosylated oligosaccharides obtained from thecompositions is determined by using MALDI-TOF analysis (e.g., see Table6, below). According to one embodiment, the percentage ofnon-fucosylated oligosaccharides is determined by measuring thepercentage of G0-Fuc (m/z 2000) released from the anti-BR3 antibodycomposition. According to another embodiment, the underfucosylatedcomposition is selected from the group consisting of 21-100%, 22-100%,23-100%, 24-100%, 25-100%, 26-100%, 27-100%, 28-100%, 29-100%, 30-100%,31-100%, 32-100%, 33-100%, 34-100%, 35-100%, 36-100%, 37-100%, 38-100%,39-100%, 40-100%, 41-100%, 42-100%, 43-100%, 44-100%, 45-100%, 46-100%,47-100%, 48-100%, 49-100%, 50-100%, 51-100%, 52-100%, 53-100%, 54-100%,55-100%, 55-100%, 56-100%, 57-100%, 58-100%, 59-100%, 60-100%, 61-100%,62-100%, 63-100%, 64-100%, 65-100%, 66-100%, 67-100%, 68-100%, 69-100%,70-100%, 71-100%, 72-100%, 73-100%, 74-100%, 75-100%, 76-100%, 77-100%,78-100%, 79-100%, 80-100%, 81-100%, 82-100%, 83-100%, 84-100%, 85-100%,86-100%, 87-100%, 88-100%, 89-100%, 90-100%, 91-100%, 92-100%, 93-100%,94-100%, 95-100%, 96-100%, 97-100%, 98-100% and 99-100% of the N-linkedoligosaccharide molecules released from the Fc region of the anti-BR3antibody composition comprise a mature core carbohydrate structure whichlacks fucose.

By “afucosylated” (AF) or “non-fucosylated” is meant the state of amolecule wherein the molecule includes a mature core carbohydratestructure which lacks a fucose. Examples of an afucosylated stateinclude G0-F (also known as G0-Fuc), G1-F, or G2-F (see FIG. 7). Forexample, an afucosylated antibody may have a fucose lacking from acarbohydrate on one heavy chain or both heavy chains of the antibodymolecule. Methods for determining the fucosylation state of a moleculeinclude capillary electrophoresis (see for example, Ragu, AnalyticalBiochemistry 283:125-132 (2000)) and mass spectrometry (e.g.,MALDI-TOF). A composition can include antibodies that are anywhere from0 to 100% afucosylated. In one embodiment, a composition can includefrom 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, or 19% of the antibodies in an afucosylated state. Inanother embodiment, a composition can include up to 100% of theantibodies in an afucosylated state.

A “bisecting GlcNAc” is a GlcNAc residue attached to the β1,4 mannose ofthe mature core carbohydrate structure. The bisecting GlcNAc can beenzymatically attached to the mature core carbohydrate structure by aβ(1,4)-N-acetylglucosaminyltransferase III enzyme (GnTIII). CHO cells donot normally express GnTIII (Stanley et al. J. Biol. Chem.261:13370-13378 (1984)), but may be engineered to do so (Umana et al.Nature Biotech. 17:176-180 (1999)).

A glycoprotein that is “essentially free” of one or more selected sugargroups (e.g. bisecting GlcNAc, one or more galactose residues, or one ormore sialic acid residues) is generally produced in a host cell that isdefective in the addition of the selected sugar group(s) to the maturecore carbohydrate structure, such that about 90-100% of the glycoproteinin a composition will lack the selected sugar group(s) attached to themature core carbohydrate structure.

A “glycosidase” is an enzyme involved in the biosynthesis ofasparagine-linked (N-linked) glycoproteins. A “trimming” enzyme is onewhich removes oligosaccharide(s), whereas a “transferase” addsoligosaccharide(s). Examples of glycosidases include trimmingglucosidases such as glucosidase I and glucosidase II; trimmingmannosidases such as rough endoplasmic reticulum mannosidase (rERmannosidase), mannosidase IA, mannosidase IB and mannosidase II; as wellas transferases such as glycosyl transferases, e.g.β(1,4)-N-acetylglucosaminyltransferase III (GnT III), Gal-transferases,sialic-acid-transferases and fuc-transferases.

A “glycosidase inhibitor” refers to a compound or composition whichreduces or prevents N-linked oligosaccharide processing by one or moreglycosidase(s). Examples include, nojirimycin, 1-deoxynojirimycin (dNM),N-Methyl-1-deoxy-nojirimycin (M-dNM), castanospermine, bromoconduritol,1-deoxymannojirimycin (dMM), australine, MDL, lentiginosine, andSwainsonine (Sw). Glycosidase inhibitors are reviewed in Fuhrmann et al.Biochim. Biophys. Acta 825:95-110 (1985); Kaushal and Elbein, Methods inEnzym. 230:316-329 (1994); and Elbein, A. FASEB 5:3055-3063 (1991).

“Lec13” refers to the lectin-resistant Chinese Hamster Ovary (CHO)mutant cell line which displays a defective fucose metabolism andtherefore has a diminished ability to add fucose to complexcarbohydrates. That cell line is described in Ripka and Stanley, SomaticCell & Molec. Gen. 12(1):51-62 (1986); and Ripka et al. Arch. Biochem.Biophys. 249(2):533-545 (1986) and is available from the Albert EinsteinCollege of Medicine of Yeshiva University, Bronx, N.Y. Lec13 cells arebelieved lack the transcript for GDP-D-mannose-4,6-dehydratase, a keyenzyme for fucose metabolism. Ohyama et al. J. Biol. Chem.273(23):14582-14587 (1988). GDP-D-mannose-4,6-dehydratase generatesGDP-mannose-4-keto-6-D-deoxymannose from GDP-mannose, which is thenconverted by the FX protein to GDP-L-fucose. Expression of fucosylatedoligosaccharides is dependent on the GDP-L-fucose donor substrates andfucosyltransferase(s).

A “fucosyltransferase” is an enzyme that adds one or more fucose(s) to aglycoprotein. Examples include α1,6-fucosyltransferase, FucTI, FucTII,FucTIII, FucTIV, FucTV, FucTVI and FucTVII. Porcine and humanα1,6-fucosyltransferases are described in Uozumi et al. J. Biol. Chem.271:27810-27817 (1996), and Yanagidani et al. J. Biochem. 121:626-632(1997), respectively.

A “sialyltransferase” is an enzyme that adds one or more sialic acidresidue(s) to a glycoprotein. An α2,3 sialytransferase can add sialicacid residue(s) to galactose residue(s) attached to a mature corecarbohydrate structure.

A “galactotransferase” is an enzyme that adds one or more galactoseresidue(s) to a glycoprotein. A β1,4-galactosyltransferase can addgalactose residue(s) to the mature core carbohydrate structure.

The term “Fc region-containing glycoprotein” refers to a glycoprotein,such as an antibody or immunoadhesin, which comprises an Fc region.

The term “Fc region” is used to define a C-terminal region of animmunoglobulin heavy chain. The “Fc region” may be a native sequence Fcregion or a variant Fc region. Although the boundaries of the Fc regionof an immunoglobulin heavy chain might vary, the human IgG heavy chainFc region is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheFc region of an immunoglobulin generally comprises two constant domains,CH2 and CH3.

The terms “BAFF,” “BAFF polypeptide,” “TALL-1,” “TALL-1 polypeptide,” or“BLyS” when used herein encompass “native sequence BAFF polypeptides”and “BAFF variants”. “BAFF” is a designation given to those polypeptideswhich are encoded by any one of the amino acid sequences of SEQ IDNO:143 or SEQ ID NO:144 and homologs and fragments and variants thereof,which have the biological activity of the native sequence BAFF. Abiological activity of BAFF can be selected from the group consisting ofpromoting B cell survival, promoting B cell maturation and binding toBR3, BCMA or TACI. Variants of BAFF will preferably have at least 80% orany successive integer up to 100% including, more preferably, at least90%, and even more preferably, at least 95% amino acid sequence identitywith a native sequence of a BAFF polypeptide. A “native sequence” BAFFpolypeptide comprises a polypeptide having the same amino acid sequenceas the corresponding BAFF polypeptide derived from nature. For example,BAFF exists in a soluble form following cleavage from the cell surfaceby furin-type proteases. Such native sequence BAFF polypeptides can beisolated from nature or can be produced by recombinant and/or syntheticmeans. The term “native sequence BAFF polypeptide” specificallyencompasses naturally-occurring truncated or secreted forms (e.g., anextracellular domain sequence), naturally-occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants ofthe polypeptide. The term “BAFF” includes those polypeptides describedin Shu et al., J. Leukocyte Biol., 65:680 (1999); GenBank Accession No.AF136293; WO98/18921 published May 7, 1998; EP 869,180 published Oct. 7,1998; WO98/27114 published Jun. 25, 1998; WO99/12964 published Mar. 18,1999; WO99/33980 published Jul. 8, 1999; Moore et al., Science,285:260-263 (1999); Schneider et al., J. Exp. Med., 189:1747-1756(1999); Mukhopadhyay et al., J. Biol. Chem., 274:15978-15981 (1999).

The term “BAFF antagonist” as used herein is used in the broadest sense,and includes any molecule that (1) binds a native sequence BAFFpolypeptide or binds a native sequence BR3 polypeptide to partially orfully block BR3 interaction with BAFF polypeptide, and (2) partially orfully blocks, inhibits, or neutralizes native sequence BAFF signaling.Native sequence BAFF polypeptide signaling promotes, among other things,B cell survival and B cell maturation. The inhibition, blockage orneutralization of BAFF signaling results in, among other things, areduction in the number of B cells. A BAFF antagonist according to thisinvention will partially or fully block, inhibit, or neutralize one ormore biological activities of a BAFF polypeptide, in vitro or in vivo.In one embodiment, a biologically active BAFF potentiates any one or anycombination of the following events in vitro or in vivo: an increasedsurvival of B cells, an increased level of IgG and/or IgM production, orstimulated B cell proliferation.

The term “TACI antagonist” as used herein is used in the broadest sense,and includes any molecule that (1) binds a native sequence BAFFpolypeptide or binds a native sequence TACI polypeptide to partially orfully block TACI interaction with BAFF polypeptide, and (2) partially orfully blocks, inhibits, or neutralizes native sequence BAFF signaling.

The term “BCMA antagonist” as used herein is used in the broadest sense,and includes any molecule that (1) binds a native sequence BAFFpolypeptide or binds a native sequence BCMA polypeptide to partially orfully block BCMA interaction with BAFF polypeptide, and (2) partially orfully blocks, inhibits, or neutralizes native sequence BAFF signaling.

As mentioned above, a BAFF antagonist can function in a direct orindirect manner to partially or fully block, inhibit or neutralize BAFFsignaling, in vitro or in vivo. For instance, the BAFF antagonist candirectly bind BAFF. For example, anti-BAFF antibodies that bind within aregion of human BAFF comprising residues 162-275 and/or a neighboringresidue of a residue selected from the group consisting of 162, 163,206, 211, 231, 233, 264 and 265 of human BAFF such that the antibodysterically hinders BAFF binding to BR3 is contemplated. In anotherexample, a direct binder is a polypeptide comprising the extracellulardomain of a BAFF receptor such as TACI, BR3 and BCMA, or comprising theboxed minimal region of the ECDs (corresponding to residues 19-35 ofhuman BR3). Alternatively, the BAFF antagonist can bind an extracellulardomain of a native sequence BR3 at its BAFF binding region to partiallyor fully block, inhibit or neutralize BAFF binding to BR3 in vitro, insitu, or in vivo. For example, such indirect antagonist is an anti-BR3antibody that binds in a region of BR3 comprising residues 23-38 ofhuman BR3 or a neighboring region of those residues such that binding ofhuman BR3 to BAFF is sterically hindered. Other examples of BAFF bindingFc proteins that can be BAFF antagonists can be found in WO 02/66516, WO00/40716, WO 01/87979, WO 03/024991, WO 02/16412, WO 02/38766, WO02/092620 and WO 01/12812. BAFF antagonists include BAFF-bindingsequences listed in FIG. 20 of WO 02/24909 and those described in WO2003/024991, WO 02/092620, fragments of those sequences that bind BAFF,and fusion proteins comprising those sequences (e.g., Fc fusionproteins).

The terms “BR3”, “BR3 polypeptide” or “BR3 receptor” when used hereinencompass “native sequence BR3 polypeptides” and “BR3 variants” (whichare further defined herein). “BR3” is a designation given to thosepolypeptides comprising any one of SEQ ID NOs:145-149 and variants orfragments thereof. The BR3 polypeptides of the invention can be isolatedfrom a variety of sources, such as from human tissue types or fromanother source, or prepared by recombinant and/or synthetic methods. Theterm BR3, includes the BR3 polypeptides described in WO 02/24909 and WO03/14294.

A “native sequence” BR3 polypeptide comprises a polypeptide having thesame amino acid sequence as the corresponding BR3 polypeptide derivedfrom nature. Such native sequence BR3 polypeptides can be isolated fromnature or can be produced by recombinant and/or synthetic means. Theterm “native sequence BR3 polypeptide” specifically encompassesnaturally-occurring truncated, soluble or secreted forms (e.g., anextracellular domain sequence), naturally-occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants ofthe polypeptide. The BR3 polypeptides of the invention include the BR3polypeptide comprising or consisting of the contiguous sequence of aminoacid residues 1 to 184 of a human BR3.

A BR3 “extracellular domain” or “ECD” refers to a form of the BR3polypeptide which is essentially free of the transmembrane andcytoplasmic domains. ECD forms of BR3 include those comprising any oneof amino acids 1 to 77, 2 to 62, 2-71, 1-61, 8-71, 17-42, 19-35 or 2-63of BR3.

“BR3 variant” means a BR3 polypeptide having at least about 60% aminoacid sequence identity with the residues 19-35 of BR3ECD and binds anative sequence BAFF polypeptide. See Gordon, N. C., et al., (2003)Biochemistry 42:5977-5983). Optionally, the BR3 variant includes asingle cysteine rich domain. Such BR3 variant polypeptides include, forinstance, BR3 polypeptides wherein one or more amino acid residues areadded, or deleted, at the N- and/or C-terminus, as well as within one ormore internal domains, of the full-length amino acid sequence. Fragmentsof the BR3 ECD that bind a native sequence BAFF polypeptide are alsocontemplated. According to an embodiment, a BR3 variant polypeptide willhave at least about 65% amino acid sequence identity, at least about 70%amino acid sequence identity, at least about 75% amino acid sequenceidentity, at least about 80% amino acid sequence identity, at leastabout 80% amino acid sequence identity, at least about 85% amino acidsequence identity, at least about 90% amino acid sequence identity, atleast about 95% amino acid sequence identity, at least about 98% aminoacid sequence identity or at least about 99% amino acid sequenceidentity in that portion corresponding to residues 19-35 of human BR3.

The term “antibody” is used in the broadest sense and specificallycovers, for example, monoclonal antibodies, polyclonal antibodies,antibodies with polyepitopic specificity, single chain antibodies,multi-specific antibodies and fragments of antibodies. According to someembodiments, a polypeptide of this invention is fused into an antibodyframework, for example, in the variable domain or in a CDR such that theantibody can bind to and inhibit BAFF binding to BR3 or BAFF signaling.The antibodies comprising a polypeptide of this invention can bechimeric, humanized, or human. The antibodies comprising a polypeptideof this invention can be an antibody fragment. Such antibodies andmethods of generating them are described in more detail below.Alternatively, an antibody of this invention can be produced byimmunizing an animal with a polypeptide of this invention. Thus, anantibody directed against a polypeptide of this invention iscontemplated.

As used herein, the terms “anti-BR3” and “BR3 binding” are usedinterchangeably and indicate that the antibody or polypeptide binds aBR3 polypeptide. Preferably, the anti-BR3 antibody binds to an epitopeon a BR3 polypeptide having an amino acid sequence selected from thegroup consisting of SEQ ID NO:145-149 and does not bind to human TACI orhuman BCMA. Preferably, the anti-BR3 antibody binds a human BR3extracellular domain sequence with an apparent Kd value of 500 nM orless, 100 nM or less, 50 nM or less, 10 nM or less, 5 nM or less or 1 nMor less as a Fab in a BIAcore Assay at 25° C. According to oneembodiment, the antibody or polypeptide binds to BR3 with an apparent Kdbetween 0.001 pM and 500 nM.

“Antagonistic anti-BR3 antibodies” according to this invention refer toantibodies that bind a BR3 polypeptide and inhibit BR3 signalling (e.g,inhibit BR3 related B cell proliferation, B cell survival or both B cellproliferation and survival).

“Agonistic anti-BR3 antibodies” according to this invention refer toantibodies that bind a BR3 polypeptide and stimulate BR3 signalling(e.g., BR3-related B cell proliferation, B cell survival or both B cellproliferation and survival).

The “CD20” antigen is a non-glycosylated, transmembrane phosphoproteinwith a molecular weight of approximately 35 kD that is found on thesurface of greater than 90% of B cells from peripheral blood or lymphoidorgans. CD20 is expressed during early pre-B cell development andremains until plasma cell differentiation; it is not found on human stemcells, lymphoid progenitor cells or normal plasma cells. CD20 is presenton both normal B cells as well as malignant B cells. Other names forCD20 in the literature include “B-lymphocyte-restricted differentiationantigen” and “Bp35.” The CD20 antigen is described in, for example,Clark and Ledbetter, Adv. Can. Res. 52:81-149 (1989) and Valentine etal. J. Biol. Chem. 264(19):11282-11287 (1989).

CD20 binding antibody and anti-CD20 antibody are used interchangeablyherein and encompass all antibodies that bind CD20 with sufficientaffinity such that the antibody is useful as a therapeutic agent intargeting a cell expressing the antigen, and do not significantlycross-react with other proteins such as a negative control protein inthe assays described below. Bispecific antibodies wherein one arm of theantibody binds CD20 are also contemplated. Also encompassed by thisdefinition of CD20 binding antibody are functional fragments of thepreceding antibodies. The CD20 binding antibody will bind CD20 with a Kdof <10 nM. In preferred embodiments, the binding is at a Kd of <7.5 nM,more preferably <5 nM, even more preferably at between 1-5 nM, mostpreferably, <1 nM.

Examples of antibodies which bind the CD20 antigen include: “C2B8” whichis now called “Rituximab” (“Rituxane®”) (U.S. Pat. No. 5,736,137,expressly incorporated herein by reference); the yttrium-[90]-labeled2B8 murine antibody designated “Y2B8” or “Ibritumomab Tiuxetan” ZEVALIN®(U.S. Pat. No. 5,736,137, expressly incorporated herein by reference);murine IgG2a “B1,” also called “Tositumomab,” (Beckman Coulter)optionally labeled with ¹³¹I to generate the “131I-B1” antibody (iodineI131 tositumomab, BEXXAR™) (U.S. Pat. No. 5,595,721, expresslyincorporated herein by reference); murine monoclonal antibody “1F5”(Press et al. Blood 69(2):584-591 (1987) and variants thereof including“framework patched” or humanized IFS (WO03/002607, Leung, S.); ATCCdeposit HB-96450); murine 2H7 and chimeric 2H7 antibody (U.S. Pat. No.5,677,180, expressly incorporated herein by reference); humanized 2H7;huMax-CD20 (Genmab, Denmark); AME-133 (Applied Molecular Evolution); A20antibody or variants thereof such as chimeric or humanized A20 antibody(cA20, hA20, respectively) (US 2003/0219433, Immunomedics); andmonoclonal antibodies L27, G28-2, 93-1B3, B-C1 or NU-B2 available fromthe International Leukocyte Typing Workshop (Valentine et al., In:Leukocyte Typing III (McMichael, Ed., p. 440, Oxford University Press(1987)).

The terms “rituximab” or “Rituxan®” herein refer to the geneticallyengineered chimeric murine/human monoclonal antibody directed againstthe CD20 antigen and designated “C2B8” in U.S. Pat. No. 5,736,137expressly incorporated herein by reference, including fragments thereofwhich retain the ability to bind CD20 .

In a specific embodiment, the anti-CD20 antibodies bind human andprimate CD20. In specific embodiments, the antibodies that bind CD20 arehumanized or chimeric. CD20 binding antibodies include rituximab(Rituxan®), m2H7 (murine 2H7), hu2H7 (humanized 2H7) and all itsfunctional variants, including without limitation, hu2H7.v16 (v standsfor version), v31, v73, v75, as well as fucose deficient variants, andother 2H7 variants described in WO2004/056312. Unless indicated, thesequences disclosed herein of the humanized 2H7v.16 and variants thereofare of the mature polypeptide, i.e., without the leader sequence.

Patents and patent publications concerning CD20 antibodies include U.S.Pat. Nos. 5,776,456, 5,736,137, 5,843,439, 6,399,061, and 6,682,734, aswell as US patent appln nos. US 2002/0197255A1, US 2003/0021781A1, US2003/0082172 A1, US 2003/0095963 A1, US 2003/0147885 A1 (Anderson etal.); U.S. Pat. No. 6,455,043B1 and WO00/09160 (Grillo-Lopez, A.);WO00/27428 (Grillo-Lopez and White); WO00/27433 (Grillo-Lopez andLeonard); WO00/44788 (Braslawsky et al.); WO01/10462 (Rastetter, W.);WO01/10461 (Rastetter and White); WO01/10460 (White and Grillo-Lopez);US2001/0018041A1, US2003/0180292A1, WO01/34194 (Hanna and Hariharan); USappln no. US2002/0006404 and WO02/04021 (Hanna and Hariharan); US applnno. US2002/0012665 A1 and WO01/74388 (Hanna, N.); US appln no. US2002/0058029 A1 (Hanna, N.); US appln no. US 2003/0103971 A1 (Hariharanand Hanna); US appln no. US2002/0009444A1, and WO01/80884 (Grillo-Lopez,A.); WO01/97858 (White, C.); US appln no. US2002/0128488A1 andWO02/34790 (Reff, M.); WO02/060955 (Braslawsky et al.); WO2/096948(Braslawsky et al.); WO02/079255 (Reff and Davies); U.S. Pat. No.6,171,586B1, and WO98/56418 (Lam et al.); WO98/58964 (Raju, S.);WO99/22764 (Raju, S.); WO99/51642, U.S. Pat. No. 6,194,551B1, U.S. Pat.No. 6,242,195B1, U.S. Pat. No. 6,528,624B1 and U.S. Pat. No. 6,538,124(Idusogie et al.); WO00/42072 (Presta, L.); WO00/67796 (Curd et al.);WO01/03734 (Grillo-Lopez et al.); US appln no US 2002/0004587A1 andWO01/77342 (Miller and Presta); US appln no. US2002/0197256 (Grewal,I.); US Appln no. US 2003/0157108 A1 (Presta, L.); U.S. Pat. Nos.6,565,827B1, 6,090,365B1, 6,287,537B1, 6,015,542, 5,843,398, and5,595,721, (Kaminski et al.); U.S. Pat. Nos. 5,500,362, 5,677,180,5,721,108, 6,120,767, 6,652,852B1 (Robinson et al.); U.S. Pat. No.6,410,391B1 (Raubitschek et al.); U.S. Pat. No. 6,224,866B1 andWO00/20864 (Barbera-Guillem, E.); WO01/13945 (Barbera-Guillem, E.);WO00/67795 (Goldenberg); US Appl No. US 2003/0133930 A1 and WO00/74718(Goldenberg and Hansen); WO00/76542 (Golay et al.); WO01/72333 (Wolinand Rosenblatt); U.S. Pat. No. 6,368,596B1 (Ghetie et al.); U.S. Pat.No. 6,306,393 and US Appln no. US2002/0041847 A1, (Goldenberg, D.); USAppln No. US2003/0026801A1 (Weiner and Hartmann); WO02/102312 (Engleman,E.); US Patent Application No. 2003/0068664 (Albitar et al.);WO03/002607 (Leung, S.); WO 03/049694, US2002/0009427A1, and US2003/0185796 A1 (Wolin et al.); WO03/061694 (Sing and Siegall); US2003/0219818 A1 (Bohen et al.); US 2003/0219433 A1 and WO 03/068821(Hansen et al.); US2003/0219818A1 (Bohen et al.); US2002/0136719A1(Shenoy et al.); WO2004/032828 (Wahl et al.), each of which is expresslyincorporated herein by reference. See, also, U.S. Pat. No. 5,849,898 andEP appln no. 330,191 (Seed et al.); U.S. Pat. No. 4,861,579 andEP332,865A2 (Meyer and Weiss); U.S. Pat. No. 4,861,579 (Meyer et al.);WO95/03770 (Bhat et al.); US 2003/0219433 A1 (Hansen et al.).

The CD20 antibodies can be naked antibody or conjugated to a cytotoxiccompound such as a radioisotope, or a toxin. Such antibodies include theantibody Zevalin™ which is linked to the radioisotope, Yttrium-90 (IDECPharmaceuticals, San Diego, Calif.), and Bexxar™ which is conjugated toI-131 (Corixa, Wash.). The humanized 2H7 variants include those thathave amino acid substitutions in the FR and affinity maturation variantswith changes in the grafted CDRs. The substituted amino acids in the CDRor FR are not limited to those present in the donor or acceptorantibody. In other embodiments, the anti-CD20 antibodies of theinvention further comprise changes in amino acid residues in the Fcregion that lead to improved effector function including enhanced CDCand/or ADCC function and B-cell killing (also referred to herein asB-cell depletion). In particular, three mutations have been identifiedfor improving CDC and ADCC activity: S298A/E333A/K334A (also referred toherein as a triple Ala mutant or variant; numbering in the Fc region isaccording to the EU numbering system; Kabat et al., supra) as described(Idusogie et al., supra (2001); Shields et al., supra).

Other anti-CD20 antibodies of the invention include those havingspecific changes that improve stability. In one embodiment, the chimericanti-CD20 antibody has murine V regions and human C region. One suchspecific chimeric anti-CD20 antibody is Rituxan® (Rituximab®; Genentech,Inc.). Rituximab and hu2H7 can mediate lysis of B-cells through bothcomplement-dependent cytotoxicity (CDC) and antibody-dependent cellularcytotoxicity (ADCC). Antibody variants with altered Fc region amino acidsequences and increased or decreased C1q binding capability aredescribed in U.S. Pat. No. 6,194,551B1 and WO99/51642. The contents ofthose patent publications are specifically incorporated herein byreference. See, also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

Inhibitors of Apoptosis (IAP) refers to a family of proteins thatinhibit apoptosis (Deveraux, et al., (1999) Genes Dev 13(3):239-252).Examples of IAPs includes melanoma IAP (ML-IAP) and human X-chromosomelinked IAP (XIAP) cellular IAP 1 (cIAP-1), and cellular IAP 2 (cIAP-2),which inhibit caspase 3, caspase 7 and caspase 9 activity (Deveraux etal., J Clin Immunol (1999), 19:388-398; Deveraux et al., (1998) EMBO J.17, 2215-2223; Vucic et al., (2000) Current Bio 10:1359-1366).

Examples of inhibitors of IAP (IAP inhibitors) includes antisenseoligonucleotides directed against XIAP, clAP-1, cIAP-2 or ML-IAP,Smac/DIABLO-derived peptides or other molecules that block theinteraction between IAPs and their caspases, and molecules that inhibitIAP-mediated suppression of caspase activity (Sasaki et al, Cancer Res.,2000, 60(20):5659; Lin et al, Biochem J., 2001, 353:299; Hu et al, Clin.Cancer Res., 2003, 9(7):2826; Arnt et al, 1. Biol. Chem., 2002,277(46):44236; Fulda et al, Nature Med., 2002, 8(8):808; Guo et al,Blood, 2002, 99(9):3419; Vucic et al, J. Biol. Chem., 2002,277(14):12275; Yang et al, Cancer Res., 2003, 63(4):831); WO2005/097791, WO 2005/094818, US 2005/0197403 and U.S. Pat. No.6,673,917).

A “B cell surface marker” or “B cell surface antigen” herein is anantigen expressed on the surface of a B cell which can be targeted withan antagonist which binds thereto. Exemplary B cell surface markersinclude, but are not limited to, CD10, CD19, CD20, CD21, CD22, CD23,CD24, CD37, CD40, CD52, D53, CD72, CD73, CD74, CDw75, CDw76, CD77,CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85, CD86, CD180(RP105), FcRH2 (IRTA4), CD79A, C79B, CR2, CCR6, CD72, P2×5, HLA-DOB,CXCR5 (BLR1), FCER2, BR3 (aka BAFF-R), TACI, BTLA, NAG14 (aka LRRC4),SLGC16270 (ala LOC283663), FcRH1 (IRTA5), FcRH5 (IRTA2), ATWD578 (akaMGC15619), FcRH3 (IRTA3), FcRH4 (IRTA1), FcRH6 (aka LOC343413) and BCMA(aka TNFRSFI7), HLA-DO, HLA-Dr10 and MHC ClassII.

According to a preferred embodiment, the antibodies of this invention donot include the 9A antibody and the 2.1 antibody deposited and describedin WO 02/24909.

According to one preferred embodiment, the “apparent Kd” or “apparent Kdvalue” as used herein is in one preferred embodiment is measured bysurface plasmon resonance such as by performing a BIAcore® assay. In onepreferred embodiment, an apparent Kd value for a BR3-binding antibody ofthis invention is measured by performing surface plasmon resonancewherein either a BR3 ECD is immobilized on a sensor chip and an anti-BR3antibody in Fab form is flowed over the BR3 ECD-immobilized chip or ananti-BR3 antibody in IgG form is immobilized on a sensor chip and a BR3ECD is flowed over the IgG-immobilized sensor chip, e.g., as describedin Example 8 herein. According to one preferred embodiment, the sensorchips are immobilized with protein such that there is approximately 10response units (RU) of coupled protein on a chip. In another preferredembodiment, an apparent Kd value for an FeRn-binding antibody of thisinvention is measured by performing surface plasmon resonance wherein aFcRn polypeptide is immobilized to a sensor chip and an antibody isflowed over the chip, e.g., as described in Example 16.

A “functional epitope” according to this invention refers to amino acidresidues of an antigen that contribute energetically to the binding ofan antibody. Mutation of any one of the energetically contributingresidues of the antigen (for example, mutation of wild-type BR3 byalanine or homolog mutation) will disrupt the binding of the antibody tothe antigen. In one preferred embodiment of this invention, a residuethat is comprised within the functional epitope on an anti-BR3 antibodycan be determined by shot-gun alanine scanning using phage displayingala mutants of BR3 or a portion thereof (e.g, the extracellular domainor residues 17-42 if desired region of study). According to onepreferred embodiment, the functional epitope is determined according tothe procedure described in Example 9.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vregions mediate antigen binding and define specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the 110-amino acid span of the variabledomains. Instead, the V domains consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting abeta-sheet configuration, connected by three hypervariable regions,which form loops connecting, and in some cases forming part of, thebeta-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V_(L), and aroundabout 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the V_(H) (Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991))and/or those residues from a “hypervariable loop” (e.g. residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the V_(L), and 26-32 (H1), 52A-55(H2) and 96-101 (H3) in the V_(H) (Chothia and Lesk J. Mol. Biol.196:901-917 (1987)).

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in theVL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3)in the VH. The variable domain residues are numbered according to Kabatet al., supra for each of these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined. For example,light chain framework 1 (LC-FR1), framework 2 (LC-FR2), framework 3(LC-FR3) and framework 4 (LC-FR4) region comprise residues numbered1-23, 35-49, 57-88 and 98-107 of an antibody (Kabat numbering system),respectively. In another example, heavy chain framework 1 (HC-FR1),heavy chain framework 2 (HC-FR2), heavy chain framework 3 (HC-FR3) andheavy chain framework 4 (HC-FR4) comprise residues 1-25, 36-48, 66-92and 103-113, respectively, of an antibody (Kabat numbering system).

As referred to herein, the “consensus sequence” or consensus V domainsequence is an artificial sequence derived from a comparison of theamino acid sequences of known human immunoglobulin variable regionsequences. Based on these comparisons, recombinant nucleic acidsequences encoding the V domain amino acids that are a consensus of thesequences derived from the human and the human H chain subgroup III Vdomains were prepared. The consensus V sequence does not have any knownantibody binding specificity or affinity.

The term “monoclonal antibody” as used herein refers to an antibody froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical and/orbind the same epitope(s), except for possible variants that may ariseduring production of the monoclonal antibody, such variants generallybeing present in minor amounts. Such monoclonal antibody typicallyincludes an antibody comprising a polypeptide sequence that binds atarget, wherein the target-binding polypeptide sequence was obtained bya process that includes the selection of a single target bindingpolypeptide sequence from a plurality of polypeptide sequences. Forexample, the selection process can be the selection of a unique clonefrom a plurality of clones, such as a pool of hybridoma clones, phageclones or recombinant DNA clones. It should be understood that theselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity, themonoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by a variety of techniques,including the hybridoma method (e.g., Kohler et al., Nature, 256:495(1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681, (Elsevier, N.Y.,1981), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),phage display technologies (see, e.g., Clackson et al., Nature,352:624-628 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991);Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol.Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA101(34):12467-12472 (2004); and Lee et al. J. Immunol. Methods284(1-2):119-132 (2004) and technologies for producing human orhuman-like antibodies from animals that have parts or all of the humanimmunoglobulin loci or genes encoding human immunoglobulin sequences(see, e.g., WO98/24893, WO/9634096, WO/9633735, and WO/91 10741,Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Yearin Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669(all of GenPharm); 5,545,807; WO 97/17852, U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks etal., Bio/Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al.,Nature Biotechnology, 14: 845-851 (1996); Neuberger, NatureBiotechnology, 14: 826 (1996); and Lonberg and Huszar, Intern. Rev.Immunol., 13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while portions of the remainderof the chain(s) is identical with or homologous to correspondingsequences in antibodies derived from another species or belonging toanother antibody class or subclass, as well as fragments of suchantibodies, so long as they exhibit the desired biological activity(U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). Methods of making chimeric antibodies are known inthe art.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin. Insome embodiments, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from acomplementarity-determining region (CDR) of the recipient are replacedby residues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are generally made to further refine andmaximize antibody performance. Typically, the humanized antibody willcomprise substantially all of at least one variable domain, in which allor substantially all of the hypervariable loops derived from a non-humanimmunoglobulin and all or substantially all of the FR regions arederived from a human immunoglobulin sequence although the FR regions mayinclude one or more amino acid substitutions to, e.g., improve bindingaffinity. In some embodiments, the number of these amino acidsubstitutions in the FR are typically no more than 6 in the H chain, andin the L chain, no more than 3. In one preferred embodiment, thehumanized antibody will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin or a human consensus constant sequence. For furtherdetails, see Jones et al., Nature, 321:522-525 (1986); Reichmann et al.,Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992). The humanized antibody includes a PRIMATIZED® antibodywherein the antigen-binding region of the antibody is derived from anantibody produced by, e.g., immunizing macaque monkeys with the antigenof interest. Methods of making humanized antibodies are known in theart.

Human antibodies can also be produced using various techniques known inthe art, including phage-display libraries. Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991).The techniques of Cole et al. and Boerner et al. are also available forthe preparation of human monoclonal antibodies. Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner etal., J. Immunol., 147(1):86-95 (1991). See also, Lonberg and Huszar,Int. Rev. Immunol. 13:65-93 (1995). PCT publications WO 98/24893; WO92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S.Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Functional fragments” of the BR3 binding antibodies of the inventionare those fragments that retain binding to BR3 with substantially thesame affinity as the intact full chain molecule from which they arederived and are active in at least one assay selected from the groupconsisting of depletion of B cells, inhibition of B cell proliferationor inhibition of BAFF binding to BR3 as measured by in vitro or in vivoassays such as those described herein.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation. A “native sequence Fc region” comprises an amino acidsequence identical to the amino acid sequence of an Fc region found innature. Examples of Fc sequences are described in SEQ ID NOs:.133,135-141 and include a native sequence human IgG1 Fc region (non-A and Aallotypes, SEQ ID NO:133 and 135, respectively); native sequence humanIgG2 Fc region (SEQ ID NO:136); native sequence human IgG3 Fc region(SEQ ID NO:137); and native sequence human IgG4 Fc region (SEQ IDNO:138) as well as naturally occurring variants thereof. Examples ofnative sequence murine Fc regions are described in SEQ ID NOs:139-142(IgG1, IgG2a, IgG2b, IgG3, respectively).

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one“amino acid modification” as herein defined. Preferably, the variant Fcregion has at least one amino acid substitution compared to a nativesequence Fc region or to the Fc region of a parent polypeptide, e.g.from about one to about ten amino acid substitutions, and preferablyfrom about one to about five amino acid substitutions in a nativesequence Fc region or in the Fc region of the parent polypeptide. In oneembodiment, the variant Fc region herein will possess at least about 80%homology, at least about 85% homology, at least about 90% homology, atleast about 95% homology or at least about 99% homology with a nativesequence Fc region (e.g., SEQ ID NO: 133). According to anotherembodiment, the variant Fc region herein will possess at least about 80%homology, at least about 85% homology, at least about 90% homology, atleast about 95% homology or at least about 99% homology with an Fcregion of a parent polypeptide.

“Percent (%) amino acid sequence identity” or “homology” with respect tothe polypeptide and antibody sequences identified herein is defined asthe percentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the polypeptide beingcompared, after aligning the sequences considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2. The ALIGN-2 sequence comparison computerprogram was authored by Genentech, Inc. and the source code has beenfiled with user documentation in the U.S. Copyright Office, WashingtonD.C., 20559, where it is registered under U.S. Copyright RegistrationNo. TXU510087. The ALIGN-2 program is publicly available throughGenentech, Inc., South San Francisco, Calif. The ALIGN-2 program shouldbe compiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

The term “Fc region-comprising polypeptide” refers to a polypeptide,such as an antibody or immunoadhesin (see definitions below), whichcomprises an Fc region. The C-terminal lysine (residue 447 according tothe EU numbering system) of the Fc region may be removed, for example,during purification of the polypeptide or by recombinantly engineeringthe nucleic acid encoding the polypeptide. Accordingly, a compositioncomprising polypeptides, including antibodies, having an Fc regionaccording to this invention can comprise polypeptides populations withall K447 residues removed, polypeptide populations with no K447 residuesremoved or polypeptide populations having a mixture of polypeptides withand without the K447 residue.

Throughout the present specification and claims, the Kabat numberingsystem is generally used when referring to a residue in the variabledomain (approximately, residues 1-107 of the light chain and residues1-113 of the heavy chain) (e.g, Kabat et al., Sequences of ImmunologicalInterest. 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The “EU numbering system” or “EU index” isgenerally used when referring to a residue in an immunoglobulin heavychain constant region (e.g., the EU index reported in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991) expresslyincorporated herein by reference). Unless stated otherwise herein,references to residues numbers in the variable domain of antibodiesmeans residue numbering by the Kabat-numbering system. Unless statedotherwise herein, references to residue numbers in the constant domainof antibodies means residue numbering by the EU numbering system (e.g.,see U.S. Provisional Application No. 60/640,323, Figures for EUnumbering).

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. In one embodiment, an FcR of thisinvention is one that binds an IgG antibody (a gamma receptor) andincludes receptors of the FcγRI, FcγRII, and FcγRIII subclasses,including allelic variants and alternatively spliced forms of thesereceptors. FcγRII receptors include FcγRIIA (an “activating receptor”)and FcγRIIB (an “inhibiting receptor”), which have similar amino acidsequences that differ primarily in the cytoplasmic domains thereof.Activating receptor FcγRIIA contains an immunoreceptor tyrosine-basedactivation motif (ITAM) in its cytoplasmic domain. Inhibiting receptorFcγRIIB contains an immunoreceptor tyrosine-based inhibition motif(ITIM) in its cytoplasmic domain. (see review M. in Daëron, Annu. Rev.Immunol. 15:203-234 (1997)). The term includes allotypes, such asFcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131and/or FcγRIIA-H131. FcRs are reviewed in Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); andde Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs,including those to be identified in the future, are encompassed by theterm “FcR” herein. The term also includes the neonatal receptor, FcRn,which is responsible for the transfer of maternal IgGs to the fetus(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.24:249 (1994)).

The term “FcRn” refers to the neonatal Fc receptor (FcRn). FcRn isstructurally similar to major histocompatibility complex (MHC) andconsists of an α-chain noncovalently bound to β2-microglobulin. Themultiple functions of the neonatal Fc receptor FcRn are reviewed inGhetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays arole in the passive delivery of immunoglobulin IgGs from mother to youngand the regulation of serum IgG levels. FcRn can act as a salvagereceptor, binding and transporting pinocytosed IgGs in intact form bothwithin and across cells, and rescuing them from a default degradativepathway.

WO00/42072 (Presta) and Shields et al. J. Biol. Chem. 9(2): 6591-6604(2001) describe antibody variants with improved or diminished binding toFcRs. The contents of those publications are specifically incorporatedherein by reference.

The “CH1 domain” of a human IgG Fc region (also referred to as “C1” of“H1” domain) usually extends from about amino acid 118 to about aminoacid 215 (EU numbering system).

“Hinge region” is generally defined as stretching from Glu216 to Pro230of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regionsof other IgG isotypes may be aligned with the IgG1 sequence by placingthe first and last cysteine residues forming inter-heavy chain S—S bondsin the same positions.

The “lower hinge region” of an Fc region is normally defined as thestretch of residues immediately C-terminal to the hinge region, i.e.residues 233 to 239 of the Fc region. In previous reports, FcR bindingwas generally attributed to amino acid residues in the lower hingeregion of an IgG Fc region.

The “CH2 domain” of a human IgG Fc region (also referred to as “C2” of“H2” domain) usually extends from about amino acid 231 to about aminoacid 340. The CH2 domain is unique in that it is not closely paired withanother domain. Rather, two N-linked branched carbohydrate chains areinterposed between the two CH2 domains of an intact native IgG molecule.It has been speculated that the carbohydrate may provide a substitutefor the domain-domain pairing and help stabilize the CH2 domain. Burton,Molec. Immunol. 22:161-206 (1985).

The “CH3 domain” (also referred to as “C2” or “113” domain) comprisesthe stretch of residues C-terminal to a CH2 domain in an Fc region (i.e.from about amino acid residue 341 to the C-terminal end of an antibodysequence, typically at amino acid residue 446 or 447 of an IgG)

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include C1q binding;complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor, BCR), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g. an antibody variable domain) and can beassessed using various assays as herein disclosed, for example.

“C1q” is a polypeptide that includes a binding site for the Fc region ofan immunoglobulin. C1q together with two serine proteases, C1r and C1s,forms the complex C1, the first component of the complement dependentcytotoxicity (CDC) pathway. Human C1q can be purchased commerciallyfrom, e.g. Quidel, San Diego, Calif.

The term “binding domain” refers to the region of a polypeptide thatbinds to another molecule. In the case of an FcR, the binding domain cancomprise a portion of a polypeptide chain thereof (e.g. the alpha chainthereof) which is responsible for binding an Fc region. One usefulbinding domain is the extracellular domain of an FcR alpha chain.

A polypeptide with a variant IgG Fc with “altered” FcR binding affinityor ADCC activity is one which has either enhanced or diminished FcRbinding activity (e.g, FcγR or FcRn) and/or ADCC activity compared to aparent polypeptide or to a polypeptide comprising a native sequence Fcregion. The variant Fc which “exhibits increased binding” to an FcRbinds at least one FcR with higher affinity (e.g., lower apparent Kd orIC50 value) than the parent polypeptide or a native sequence IgG Fc.According to some embodiments, the improvement in binding compared to aparent polypeptide is about 3 fold, preferably about 5, 10, 25, 50, 60,100, 150, 200, up to 500 fold, or about 25% to 1000% improvement inbinding. The polypeptide variant which “exhibits decreased binding” toan FcR, binds at least one FcR with lower affinity (e.g, higher apparentKd or higher IC50 value) than a parent polypeptide. The decrease inbinding compared to a parent polypeptide may be about 40% or moredecrease in binding. In one embodiment, Fc variants which displaydecreased binding to an FcR possess little or no appreciable binding toan FcR, e.g., 0-20% binding to the FcR compared to a native sequence IgGFc region, e.g. as determined in the Examples herein.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 or in the Examples below may be performed.Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. PNAS (USA) 95:652-656 (1998).

The polypeptide comprising a variant Fc region which “exhibits increasedADCC” or mediates antibody-dependent cell-mediated cytotoxicity (ADCC)in the presence of human effector cells more effectively than apolypeptide having wild type IgG Fc or a parent polypeptide is one whichin vitro or in vivo is substantially more effective at mediating ADCC,when the amounts of polypeptide with variant Fc region and thepolypeptide with wild type Fc region (or the parent polypeptide) in theassay are essentially the same. Generally, such variants will beidentified using the in vitro ADCC assay as herein disclosed, but otherassays or methods for determining ADCC activity, e.g. in an animal modeletc, are contemplated. In one embodiment, the preferred variant is fromabout 5 fold to about 100 fold, e.g. from about 25 to about 50 fold,more effective at mediating ADCC than the wild type Fc (or parentpolypeptide).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), may be performed.

Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1 and WO99/51642. The contents of those patentpublications are specifically incorporated herein by reference. See,also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. According to one embodiment, the cellsexpress at least FcγRIII and perform ADCC effector function. Examples ofhuman leukocytes which mediate ADCC include peripheral blood mononuclearcells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cellsand neutrophils; with PBMCs and NK cells being preferred. The effectorcells may be isolated from a native source thereof, e.g. from blood orPBMCs as described herein.

Methods of measuring binding to FcRn are known (see, e.g., Ghetie 1997,Hinton 2004) as well as described in the Examples below. Binding tohuman FcRn in vivo and serum half life of human FcRn high affinitybinding polypeptides can be assayed, e.g, in transgenic mice ortransfected human cell lines expressing human FcRn, or in primatesadministered with the Fc variant polypeptides. In one embodiment, thepolypeptide and specifically the antibodies of the invention having avariant IgG Fc exhibits increased binding affinity for human FcRn over apolypeptide having wild-type IgG Fc, by at least 2 fold, at least 5fold, at least 10 fold, at least 50 fold, at least 60 fold, at least 70fold, at least 80 fold, at least 100 fold, at least 125 fold, at least150 fold. In a specific embodiment, the binding affinity for human FcRnis increased about 170 fold.

For binding affinity to FcRn, in one embodiment, the EC50 or apparent Kd(at pH 6.0) of the polypeptide is less than 1 uM, more preferably lessthan or equal to 100 nM, more preferably less than or equal to 10 nM. Inone embodiment, for increased binding affinity to FcγRIII (F158; i.e.low-affinity isotype) the EC50 or apparent Kd less is than or equal to10 nM, and for FcγRIII (V158; high-affinity isotype) the EC50 orapparent Kd is less than or equal to 3 nM. According to anotherembodiment, a reduction in binding of an antibody to a Fc receptorrelative to a control antibody (e.g., the Herceptin® antibody) may beconsidered significant relative to the control antibody if the ratio ofthe values of the absorbances at the midpoints of the test antibody andcontrol antibody binding curves (e.g,A_(450 nm(antibody/A450 nm(control Ab))) is less than or equal to 40%.According to another embodiment, an increase in binding of an antibodyto a Fc receptor relative to a control antibody (e.g., the Herceptin®antibody) may be considered significant relative to the control antibodyif the ratio of the values of the absorbances at the midpoints of thetest antibody and control antibody binding curves (e.g,A_(450 nm(antibody/A450 nm(control Ab))) is greater than or equal to125%. See, e.g., Example 10.

A “parent polypeptide” or “parent antibody” is a polypeptide or antibodycomprising an amino acid sequence from which the variant polypeptide orantibody arose and against which the variant polypeptide or antibody isbeing compared. Typically the parent polypeptide or parent antibodylacks one or more of the Fc region modifications disclosed herein anddiffers in effector function compared to a polypeptide variant as hereindisclosed. The parent polypeptide may comprise a native sequence Fcregion or an Fc region with pre-existing amino acid sequencemodifications (such as additions, deletions and/or substitutions).

A “fusion protein” and a “fusion polypeptide” refer to a polypeptidehaving two portions of a polypeptide sequence covalently linkedtogether. In most embodiments, each of the portions are polypeptidesequences not typically associated with each other in nature and/or havedifferent properties. The property may be a biological property, such asactivity in vitro or in vivo. The property may also be a simple chemicalor physical property, such as binding to a target molecule, catalysis ofa reaction, etc. The two portions may be linked directly by a singlepeptide bond or through a peptide linker containing one or more aminoacid residues. Generally, the two portions will be linked in readingframe with each other.

An “isolated” antibody or polypeptide is one which has been identifiedand separated and/or recovered from a component of the environment fromwhich it was produced. Contaminant components can be, e.g., materialswhich would interfere with diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In one preferred embodiment, the antibody orpolypeptide will be purified (1) to greater than 95% by weight ofantibody as determined by the Lowry method, and most preferably morethan 99% by weight, (2) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using Coomassie blue or, preferably,silver stain. Isolated antibody or polypeptide includes the antibody orpolypeptide in situ within recombinant cells since at least onecomponent of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody or polypeptide will be preparedby at least one purification step.

An “isolated” polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide-adaptors or linkers are used in accordancewith conventional practice.

“Vector” includes shuttle and expression vectors. Typically, the plasmidconstruct will also include an origin of replication (e.g., the ColE1origin of replication) and a selectable marker (e.g., ampicillin ortetracycline resistance), for replication and selection, respectively,of the plasmids in bacteria. An “expression vector” refers to a vectorthat contains the necessary control sequences or regulatory elements forexpression of the antibodies including antibody fragment of theinvention, in bacterial or eukaryotic cells. Suitable vectors aredisclosed below.

The cell that produces a BR3 binding antibody of the invention willinclude the bacterial and eukaryotic host cells into which nucleic acidencoding the antibodies have been introduced. Suitable host cells aredisclosed below.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions,” as definedherein, can be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50 C;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42 C; or (3)overnight hybridization in a solution that employs 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon spermDNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42 C, with a 10minute wash at 42 C in 0.2×SSC (sodium chloride/sodium citrate) followedby a 10 minute high-stringency wash consisting of 0.1×SSC containingEDTA at 55 C.

“Moderately stringent conditions” can be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50 C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a polypeptide fused to a “tag polypeptide.” Thetag polypeptide has enough residues to provide an epitope against whichan antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues). Polypeptides and antibodies of thisinvention that are epitope-tagged are contemplated.

“Biologically active” and “biological activity” and “biologicalcharacteristics” with respect to an anti-BR3 polypeptide or antibody ofthis invention means the antibody or polypeptide binds BR3. According toone preferred embodiment, the antibody binds human BR3 polypeptide.

In a further embodiment, an anti-BR3 polypeptide or antibody of thisinvention also has any one, any combination or all of the followingactivities: (1) binds to a human BR3 extracellular domain sequence withan apparent Kd value of 500 nM or less, 100 nM or less, 50 nM or less,10 nM or less, 5 nM or less or 1 nM or less; (2) binds to a human BR3extracellular domain sequence and binds to a rodent BR3 extracellulardomain sequence with an apparent Kd value of 500 nM or less, 100 nM orless, 50 nM or less, 10 nM or less, 5 nM or less or 1 nM or less; and(3) inhibits human BR3 binding to human BAFF. Depending on the desireduse for the antibody, the antibody can further comprise the any one ofthe following activities (1) has antibody dependent cellularcytotoxicity (ADCC) in the presence of human effector cells compared towild-type or native sequence IgG Fc; (2) has increased ADCC in thepresence of human effector cells compared to wild-type or nativesequence IgG Fc or (3) has decreased ADCC in the presence of humaneffector cells compared to wild-type or native sequence IgG Fc.According to another embodiment, an antibody of this invention binds thehuman Fc neonatal receptor (FcRn) with a higher affinity than apolypeptide or parent polypeptide having wild type or native sequenceIgG Fc.

“Biologically active” and “biological activity” and “biologicalcharacteristics” with respect to an antagonist anti-BR3 polypeptide orantibody of this invention means the antibody or polypeptide has anyone, any combination or all of the following activities: (1) inhibits Bcell proliferation; (2) inhibits B cell survival; (3) kills or depletesB cells in vivo. According to one embodiment, the depletion of B cellswhen compared to the baseline level or appropriate negative controlwhich is not treated with such anti-BR3 antibody or polypeptide is atleast 20%. According to another embodiment, the antagonistic antibodyhas antibody dependent cellular cytotoxicity (ADCC) in the presence ofhuman effector cells compared to wild-type or native sequence IgG Fc orhas increased ADCC in the presence of human effector cells compared towild-type or native sequence IgG Fc.

The amino acid sequences specifically disclosed herein are contiguousamino acid sequences unless otherwise specified.

Variations in polypeptides of this invention described herein, can bemade, for example, using any of the techniques and guidelines forconservative and non-conservative mutations. Variations can be asubstitution, deletion or insertion of one or more codons encoding thepolypeptide that results in a change in the amino acid sequence of thepolypeptide. Amino acid substitutions can be the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, such as the replacement of a leucine with a serine,i.e., conservative amino acid replacements. Insertions or deletions canoptionally be in the range of about 1 to 5 amino acids. The variationallowed can be determined by systematically making insertions, deletionsor substitutions of amino acids in the sequence and testing theresulting variants for activity exhibited by the full-length or maturenative sequence.

The term “conservative” amino acid substitution as used within thisinvention is meant to refer to amino acid substitutions which substitutefunctionally equivalent amino acids. Conservative amino acid changesresult in minimal change in the amino acid structure or function of theresulting peptide. For example, one or more amino acids of a similarpolarity act as functional equivalents and result in a silent alterationwithin the amino acid sequence of the peptide. In general, substitutionswithin a group can be considered conservative with respect to structureand function. However, the skilled artisan will recognize that the roleof a particular residue is determined by its context within thethree-dimensional structure of the molecule in which it occurs. Forexample, Cys residues may occur in the oxidized (disulfide) form, whichis less polar than the reduced (thiol) form. The long aliphatic portionof the Arg side chain can constitute a critical feature of itsstructural or functional role, and this may be best conserved bysubstitution of a nonpolar, rather than another basic residue. Also, itwill be recognized that side chains containing aromatic groups (Trp,Tyr, and Phe) can participate in ionic-aromatic or “cation-pi”interactions. In these cases, substitution of one of these side chainswith a member of the acidic or uncharged polar group may be conservativewith respect to structure and function. Residues such as Pro, Gly, andCys (disulfide form) can have direct effects on the main chainconformation, and often may not be substituted without structuraldistortions.

Conservative substitutions include the following specific substitutionsbased on the similarities in side chains and exemplary substitutions andpreferred substitutions listed below. Amino acids may be groupedaccording to similarities in the properties of their side chains (in A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers,New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Tip(W), Met (M)(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn(N), Gln (Q)(3) acidic: Asp (D), Glu (E)(4) basic: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Tip, Tyr, Phe.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The term “amino acid” within the scope of the present invention is usedin its broadest sense and is meant to include the naturally occurring Lalpha-amino acids or residues. The commonly used one and three letterabbreviations for naturally occurring amino acids are used herein(Lehninger, A. L., Biochemistry, 2d ed., pp. 71-92, (1975), WorthPublishers, New York). The term includes D-amino acids as well aschemically modified amino acids such as amino acid analogs, naturallyoccurring amino acids that are not usually incorporated into proteinssuch as norleucine, and chemically synthesized compounds havingproperties known in the art to be characteristic of an amino acid. Forexample, analogs or mimetics of phenylalanine or proline, which allowthe same conformational restriction of the peptide compounds as naturalPhe or Pro are included within the definition of amino acid. Suchanalogs and mimetics are referred to herein as “functional equivalents”of an amino acid. Other examples of amino acids are listed by Robertsand Vellaccio (The Peptides: Analysis, Synthesis, Biology,) Eds. Grossand Meiehofer, Vol. 5 p 341, Academic Press, Inc, N.Y. 1983, which isincorporated herein by reference.

Peptides synthesized by the standard solid phase synthesis techniquesdescribed here, for example, are not limited to amino acids encoded bygenes for substitutions involving the amino acids. Commonly encounteredamino acids which are not encoded by the genetic code, include, forexample, those described in International Publication No. WO 90/01940,as well as, for example, 2-amino adipic acid (Aad) for Glu and Asp;2-aminopimelic acid (Apm) for Glu and Asp; 2-aminobutyric (Abu) acid forMet, Leu, and other aliphatic amino acids; 2-aminoheptanoic acid (Ahe)for Met, Len and other aliphatic amino acids; 2-aminoisobutyric acid(Aib) for Gly; cyclohexylalanine (Cha) for Val, and Leu and Ile;homoarginine (Har) for Arg and Lys; 2,3-diaminopropionic acid (Dpr) forLys, Arg and His; N-ethylglycine (EtGly) for Gly, Pro, and Ala;N-ethylglycine (EtGly) for Gly, Pro, and Ala; N-ethylasparigine (EtAsn)for Asn, and Gin; Hydroxyllysine (Hyl) for Lys; allohydroxyllysine(AHyl) for Lys; 3-(and 4) hydroxyproline (3Hyp, 4Hyp) for Pro, Ser, andThr; allo-isoleucine (AIle) for Ile, Leu, and Val; -amidinophenylalaninefor Ala; N-methylglycine (MeGly, sarcosine) for Gly, Pro, and Ala;N-methylisoleucine (MeIle) for Ile; Norvaline (Nva) for Met and otheraliphatic amino acids; Norleucine (Nle) for Met and other aliphaticamino acids; Ornithine (Orn or Or) for Lys, Arg and His; Citrulline(Cit) and methionine sulfoxide (MSO) for Thr, Asn and Gln;-methylphenylalanine (MePhe), trimethylphenylalanine, halo (F, Cl, Br,and I)phenylalanine, triflourylphenylalanine, for Phe.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

The term “detecting” is intended to include determining the presence orabsence of a molecule or determining qualitatively or quantitatively theamount of a molecule. The term thus refers to the use of the materials,compositions, and methods of the present invention for qualitative andquantitative determinations. In general, the particular technique usedfor detection is not critical for practice of the invention.

For example, “detecting” according to the invention may includedetecting: the presence or absence of a molecule, number of cellsexpressing the polypeptide, a change in the levels of the molecule oramount of the molecule bound to a target or target bound to themolecule; a change in biological function/activity of a molecule (e.g.,ligand or receptor binding activity, intracellular signaling (such asNF-kB activation), tumor cell proliferation, B cell proliferation, orsurvival, etc.), e.g., using methods that are known in the art. In someembodiments, “detecting” may include detecting wild type levels of themolecule (e.g., mRNA or polypeptide levels). Detecting may includequantifying a change (increase or decrease) of any value between 10% and90%, or of any value between 30% and 60%, or over 100%, when compared toa control. Detecting may include quantifying a change of any valuebetween 2-fold to 10-fold, inclusive, or more e.g., 100-fold. Thus, forexample, referral to a BR3 molecule can refer to its mRNA or protein,etc.

As used herein a “BR3 molecule” as used herein refers to a moleculesubstantially identical to: a BR3 polypeptide; a nucleic acid moleculeencoding a BR3 polypeptide; as well as isoforms, fragments, analogs, orvariants of the polypeptide or the nucleic acid molecule. For example, aBR3 molecule can include an isoform, fragment, analog, or variant of aBR3 polypeptide derived from a mammal, which BR3 molecule has theability to bind BAFF.

As used herein a “BAFF molecule” as used herein refers to a moleculesubstantially identical to: a BAFF polypeptide; a nucleic acid moleculeencoding a BAFF polypeptide; as well as isoforms, fragments, analogs, orvariants of the polypeptide or the nucleic acid molecule. For example, aBAFF molecule can include an isoform, fragment, analog, or variant of aBAFF polypeptide derived from a mammal, which BAFF molecule that has theability to bind BR3.

As used herein, a subject to be treated is a mammal (e.g., human,non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat,etc.). The subject may be a clinical patient, a clinical trialvolunteer, an experimental animal, etc. The subject may be suspected ofhaving or at risk for having a cancer or immune disease, be diagnosedwith a cancer or immune disease, or be a control subject that isconfirmed to not have a cancer. Many diagnostic methods for cancer andimmune disease and the clinical delineation of cancer or immunediagnoses are known in the art. According to one preferred embodiment,the subject to be treated according to this invention is a human.

“Treating” or “treatment” or “alleviation” refers to measures, whereinthe object is to prevent or slow down (lessen) the targeted pathologiccondition or disorder or relieve some of the symptoms of the disorder.Those in need of treatment include can include those already with thedisorder as well as those prone to have the disorder or those in whomthe disorder is to be prevented. A subject or mammal is successfully“treated” for a cancer if, after receiving a therapeutic amount of apolypeptide or an antibody of the present invention, the patient showsobservable and/or measurable reduction in or absence of one or more ofthe following: reduction in the number of cancer cells or absence of thecancer cells; reduction in the tumor size; inhibition (i.e., slow tosome extent and preferably stop) of cancer cell infiltration intoperipheral organs including the spread of cancer into soft tissue andbone; inhibition (i.e., slow to some extent and preferably stop) oftumor metastasis; inhibition, to some extent, of tumor growth; and/orrelief to some extent, one or more of the symptoms associated with thespecific cancer; reduced morbidity and mortality, and improvement inquality of life issues. To the extent the polypeptides of this inventioncan prevent growth and/or kill existing cancer cells, it can becytostatic and/or cytotoxic. Reduction of these signs or symptoms mayalso be felt by the patient.

The term “therapeutically effective amount” refers to an amount of apolypeptide of this invention effective to “alleviate” or “treat” adisease or disorder in a subject. In the case of cancer, thetherapeutically effective amount of the drug may reduce the number ofcancer cells; reduce the tumor size; inhibit (i.e., slow to some extentand preferably stop) cancer cell infiltration into peripheral organs;inhibit (i.e., slow to some extent and preferably stop) tumormetastasis; inhibit, to some extent, tumor growth; and/or relieve tosome extent one or more of the symptoms associated with the cancer. Tothe extent the drug may prevent growth and/or kill existing cancercells, it may be cytostatic and/or cytotoxic.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin can be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM. For example, useful immunoadhesins according to thisinvention can be polypeptides that comprise the BAFF binding portions ofa polypeptide or BR3 binding portions of a polypeptide (e.g., a portionof a BAFF receptor excluding the transmembrane or cytoplasmic sequencesfused to an Fc region, TACI receptor extracellular domain-Fc or BCMAextracellular domain-Fc or BR3 extracellular domain-Fc). In oneembodiment, a polypeptide sequence of this invention is fused to aconstant domain of an immunoglobulin sequence.

An “immunodeficiency disease” is a disorder or condition where theimmune response is reduced (e.g., severe combined immunodeficiency(SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADAdeficiency), X-linked agammaglobulinemia (XLA). Bruton's disease,congenital agammaglobulinemia, X-linked infantile agammaglobulinemia,acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onsetagammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,transient hypogammaglobulinemia of infancy, unspecifiedhypogammaglobulinemia, agammaglobulinemia, common variableimmunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiencywith hyper IgM, selective IgA deficiency, IgG subclass deficiency (withor without IgA deficiency), antibody deficiency with normal or elevatedIgs, immunodeficiency with thymoma, Ig heavy chain deletions, kappachain deficiency, B cell lymphoproliferative disorder (BLPD), selectiveIgM immunodeficiency, recessive agammaglobulinemia (Swiss type),reticular dysgenesis, neonatal neutropenia, severe congenitalleukopenia, thymic alymphoplasia-aplasia or dysplasia withimmunodeficiency, ataxia-telangiectasia telangiectasia (cerebellarataxia, oculocutaneous telangiectasia and immunodeficiency), shortlimbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelofsyndrome-cumbined immunodeficiency with Igs, purine nucleotidephosphorylase deficiency (PNP), MHC Class II deficiency (Bare LymphocyteSyndrome) and severe combined immunodeficiency,) or conditionsassociated with an immunodeficiency, Janus Associated Kinase 3 (JAK3)deficiency, DiGeorge's syndrome (isolated T cell deficiency) andAssociated syndromes e.g., Down syndrome, chronic mucocutaneouscandidiasis, hyper-IgE syndrome, chronic granulomatous disease, partialalbinism and WHIM syndrome (warts, hypogammaglobulinemia, infection, andmyelokathexis [retention of leukocytes in a hypercellular marrow]).

An “autoimmune disease” herein is a disease or disorder arising from anddirected against an individual's own tissues or a co-segregate ormanifestation thereof or resulting condition therefrom. Examples ofautoimmune diseases or disorders include, but are not limited toarthritis (rheumatoid arthritis such as acute arthritis, chronicrheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronicinflammatory arthritis, degenerative arthritis, infectious arthritis,Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebralarthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis,arthritis chronica progrediente, arthritis deformans, polyarthritischronica primaria, reactive arthritis, and ankylosing spondylitis),inflammatory hyperproliferative skin diseases, psoriasis such as plaquepsoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of thenails, dermatitis including contact dermatitis, chronic contactdermatitis, allergic dermatitis, allergic contact dermatitis, dermatitisherpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome,urticaria such as chronic allergic urticaria and chronic idiopathicurticaria, including chronic autoimmune urticaria,polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermalnecrolysis, scleroderma (including systemic scleroderma), sclerosis suchas systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS,primary progressive MS (PPMS), and relapsing remitting MS (RRMS),progressive systemic sclerosis, atherosclerosis, arteriosclerosis,sclerosis disseminata, and ataxic sclerosis, inflammatory bowel disease(IBD) (for example, Crohn's disease, autoimmune-mediatedgastrointestinal diseases, colitis such as ulcerative colitis, colitisulcerosa, microscopic colitis, collagenous colitis, colitis polyposa,necrotizing enterocolitis, and transmural colitis, and autoimmuneinflammatory bowel disease), pyoderma gangrenosum, erythema nodosum,primary sclerosing cholangitis, episcleritis), respiratory distresssyndrome, including adult or acute respiratory distress syndrome (ARDS),meningitis, inflammation of all or part of the uvea, iritis,choroiditis, an autoimmune hematological disorder, rheumatoidspondylitis, sudden hearing loss, IgE-mediated diseases such asanaphylaxis and allergic and atopic rhinitis, encephalitis such asRasmussen's encephalitis and limbic and/or brainstem encephalitis,uveitis, such as anterior uveitis, acute anterior uveitis, granulomatousuveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterioruveitis, or autoimmune uveitis, glomerulonephritis (GN) with and withoutnephrotic syndrome such as chronic or acute glomerulonephritis such asprimary GN, immune-mediated GN, membranous GN (membranous nephropathy),idiopathic membranous GN or idiopathic membranous nephropathy, membrano-or membranous proliferative GN (MPGN), including Type I and Type II, andrapidly progressive GN, allergic conditions, allergic reaction, eczemaincluding allergic or atopic eczema, asthma such as asthma bronchiale,bronchial asthma, and auto-immune asthma, conditions involvinginfiltration of T cells and chronic inflammatory responses, chronicpulmonary inflammatory disease, autoimmune myocarditis, leukocyteadhesion deficiency, systemic lupus erythematosus (SLE) or systemiclupus erythematodes such as cutaneous SLE, subacute cutaneous lupuserythematosus, neonatal lupus syndrome (NLE), lupus erythematosusdisseminatus, lupus (including nephritis, cerebritis, pediatric,non-renal, extra-renal, discoid, alopecia), juvenile onset (Type I)diabetes mellitus, including pediatric insulin-dependent diabetesmellitus (IDDM), adult onset diabetes mellitus (Type II diabetes),autoimmune diabetes, idiopathic diabetes insipidus, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis includinglymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis,vasculitides, including vasculitis (including large vessel vasculitis(including polymyalgia rheumatica and giant cell (Takayasu's)arteritis), medium vessel vasculitis (including Kawasaki's disease andpolyarteritis nodosa), microscopic polyarteritis, CNS vasculitis,necrotizing, cutaneous, or hypersensitivity vasculitis, systemicnecrotizing vasculitis, and ANCA-associated vasculitis, such asChurg-Strauss vasculitis or syndrome (CSS)), temporal arteritis,aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia,Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemiaincluding autoimmune hemolytic anemia (AIHA), pernicious anemia (anemiaperniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA),Factor VIII deficiency, hemophilia A, autoimmune neutropenia,pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNSinflammatory disorders, multiple organ injury syndrome such as thosesecondary to septicemia, trauma or hemorrhage, antigen-antibodycomplex-mediated diseases, anti-glomerular basement membrane disease,anti-phospholipid antibody syndrome, allergic neuritis, Bechet's orBehcet's disease, Castleman's syndrome, Goodpasture's syndrome,Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome,pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus(including pemphigus vulgaris, pemphigus foliaceus, pemphigusmucus-membrane pemphigoid, and pemphigus erythematosus), autoimmunepolyendocrinopathies, Reiter's disease or syndrome, immune complexnephritis, antibody-mediated nephritis, neuromyelitis optica,polyneuropathies, chronic neuropathy such as IgM polyneuropathies orIgM-mediated neuropathy, thrombocytopenia (as developed by myocardialinfarction patients, for example), including thrombotic thrombocytopenicpurpura (TTP) and autoimmune or immune-mediated thrombocytopenia such asidiopathic thrombocytopenic purpura (ITP) including chronic or acuteITP, autoimmune disease of the testis and ovary including autoimuneorchitis and oophoritis, primary hypothyroidism, hypoparathyroidism,autoimmune endocrine diseases including thyroiditis such as autoimmunethyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto'sthyroiditis); or subacute thyroiditis, autoimmune thyroid disease,idiopathic hypothyroidism, Grave's disease, polyglandular syndromes suchas autoimmune polyglandular syndromes (or polyglandular endocrinopathysyndromes), paraneoplastic syndromes, including neurologicparaneoplastic syndromes such as Lambert-Eaton myasthenic syndrome orEaton-Lambert syndrome, stiff-man or stiff-person syndrome,encephalomyelitis such as allergic encephalomyelitis orencephalomyelitis allergica and experimental allergic encephalomyelitis(EAE), myasthenia gravis such as thymoma-associated myasthenia gravis,cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonusmyoclonus syndrome (OMS), and sensory neuropathy, multifocal motorneuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis,lupoid hepatitis, giant cell hepatitis, chronic active hepatitis orautoimmune chronic active hepatitis, lymphoid interstitial pneumonitis,bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barrésyndrome, Berger's disease (IgA nephropathy), idiopathic IgAnephropathy, linear IgA dermatosis, primary biliary cirrhosis,pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease,Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue,idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS;Lou Gehrig's disease), coronary artery disease, autoimmune ear diseasesuch as autoimmune inner ear disease (AGED), autoimmune hearing loss,opsoclonus myoclonus syndrome (OMS), polychondritis such as refractoryor relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis,scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, whichincludes monoclonal B cell lymphocytosis (e.g., benign monoclonalgammopathy and monoclonal garnmopathy of undetermined significance,MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathiessuch as epilepsy, migraine, arrhythmia, muscular disorders, deafness,blindness, periodic paralysis, and channelopathies of the CNS, autism,inflammatory myopathy, focal segmental glomerulosclerosis (FSGS),endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmunehepatological disorder, fibromyalgia, multiple endocrine failure,Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia,demyelinating diseases such as autoimmune demyelinating diseases,diabetic nephropathy, Dressler's syndrome, alopecia greata, CRESTsyndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyl), and telangiectasia), male and female autoimmuneinfertility, mixed connective tissue disease, Chagas' disease, rheumaticfever, recurrent abortion, farmer's lung, erythema multiforme,post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung,allergic granulomatous angiitis, benign lymphocytic angiitis, Alport'ssyndrome, alveolitis such as allergic alveolitis and fibrosingalveolitis, interstitial lung disease, transfusion reaction, leprosy,malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis,aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue,endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonaryfibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis,cystic fibrosis, endophthalmitis, erythema elevatum et diutinum,erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome,Felty's syndrome, flariasis, cyclitis such as chronic cyclitis,heterochronic cyclitis, iridocyclitis, or Fuch's cyclitis,Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection,echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirusinfection, rubella virus infection, post-vaccination syndromes,congenital rubella infection, Epstein-Barr virus infection, mumps,Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea,post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis,tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrineophthamopathy, chronic hypersensitivity pneumonitis,keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathicnephritic syndrome, minimal change nephropathy, benign familial andischemia-reperfusion injury, retinal autoimmunity, joint inflammation,bronchitis, chronic obstructive airway disease, silicosis, aphthae,aphthous stomatitis, arteriosclerotic disorders, aspermiogenese,autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren'scontracture, endophthalmia phacoanaphylactica, enteritis allergica,erythema nodosum leprosum, idiopathic facial paralysis, chronic fatiguesyndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearingloss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,leucopenia, mononucleosis infectiosa, traverse myelitis, primaryidiopathic myxedema, nephrosis, ophthalmia symphatica, orchitisgranulomatosa, pancreatitis, polyradiculitis acuta, pyodermagangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,infertility due to antispermatozoan antobodies, non-malignant thymoma,vitiligo, SCID and Epstein-Barr virus-associated diseases, acquiredimmune deficiency syndrome (AIDS), parasitic diseases such asLesihmania, toxic-shock syndrome, food poisoning, conditions involvinginfiltration of T cells, leukocyte-adhesion deficiency, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, diseases involving leukocyte diapedesis, multipleorgan injury syndrome, antigen-antibody complex-mediated diseases,antiglomerular basement membrane disease, allergic neuritis, autoimmunepolyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophicgastritis, sympathetic ophthalmia, rheumatic diseases, mixed connectivetissue disease, nephrotic syndrome, insulitis, polyendocrine failure,peripheral neuropathy, autoimmune polyglandular syndrome type I,adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis,dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA),hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosingcholangitis, purulent or nonpurulent sinusitis, acute or chronicsinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, aneosinophil-related disorder such as eosinophilia, pulmonary infiltrationeosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chroniceosinophilic pneumonia, tropical pulmonary eosinophilia,bronchopneumonic aspergillosis, aspergilloma, or granulomas containingeosinophils, anaphylaxis, seronegative spondyloarthritides,polyendocrine autoimmune disease, sclerosing cholangitis, sclera,episclera, chronic mucocutaneous candidiasis, Bruton's syndrome,transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome,ataxia telangiectasia, autoimmune disorders associated with collagendisease, rheumatism, neurological disease, ischemic re-perfusiondisorder, reduction in blood pressure response, vascular dysfunction,antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia,cerebral ischemia, and disease accompanying vascularization, allergichypersensitivity disorders, glomerulonephritides, reperfusion injury,reperfusion injury of myocardial or other tissues, dermatoses with acuteinflammatory components, acute purulent meningitis or other centralnervous system inflammatory disorders, ocular and orbital inflammatorydisorders, granulocyte transfusion-associated syndromes,cytokine-induced toxicity, acute serious inflammation, chronicintractable inflammation, pyelitis, pneumonocirrhosis, diabeticretinopathy, diabetic large-artery disorder, endarterial hyperplasia,peptic ulcer, valvulitis, and endometriosis.

As used herein, “B cell depletion” refers to a reduction in B celllevels in an animal or human after drug or antibody treatment, ascompared to the B cell level before treatment. B cell levels aremeasurable using well known assays such as those described in theExperimental Examples. B cell depletion can be complete or partial. Inone embodiment, the depletion of BR3 expressing B cells is at least 25%.Not to be limited by any one mechanism, possible mechanisms of B-celldepletion include ADCC, CDC, apoptosis, modulation of calcium flux or acombination of two or more of the preceding.

A “B cell surface marker” or “B cell surface antigen” herein is anantigen expressed on the surface of a B cells.

“B cell depletion agents” refers to agents that reduce peripheral Bcells by at least 25%. In another embodiment, the depletion ofperipheral B cells is at least 30%, 40%, 50%, 60%, 70%, 80% or 90%. Inone preferred embodiment, the B cell depletion agent specifically bindsto a white blood cell and not other cells types. In another embodiment,the B cell depletion agent specifically binds to a B cell and not othercell types. In one embodiment, the B cell depletion agent is anantibody. In one preferred embodiment, the antibody is a monoclonalantibody. In another embodiment, the antibody is conjugated to achemotherapeutic agent or a cytotoxic agent. Specific examples of B celldepletion agents include, but are not limited to, the aforementionedanti-CD20 antibodies.

The B cell neoplasms include Hodgkin's disease including lymphocytepredominant Hodgkin's disease (LPHD); non-Hodgkin's lymphoma (NHL);follicular center cell (FCC) lymphomas; acute lymphocytic leukemia(ALL); chronic lymphocytic leukemia (CLL); Hairy cell leukemia andBR3-positive neoplasms. The non-Hodgkins lymphoma include lowgrade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL)NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL,high grade immunoblastic NHL, high grade lymphoblastic NHL, high gradesmall non-cleaved cell NHL, bulky disease NHL, plasmacytoid lymphocyticlymphoma, mantle cell lymphoma, AIDS—related lymphoma and Waldenstrom'smacroglobulinemia. Treatment of relapses of these cancers are alsocontemplated. LPHD is a type of Hodgkin's disease that tends to relapsefrequently despite radiation or chemotherapy treatment and can becharacterized by BR3-positive malignant cells. CLL is one of four majortypes of leukemia. A cancer of mature B-cells called lymphocytes, CLL ismanifested by progressive accumulation of cells in blood, bone marrowand lymphatic tissues. Indolent lymphoma is a slow-growing, incurabledisease in which the average patient survives between six and 10 yearsfollowing numerous periods of remission and relapse.

The term “non-Hodgkin's lymphoma” or “NHL”, as used herein, refers to acancer of the lymphatic system other than Hodgkin's lymphomas. Hodgkin'slymphomas can generally be distinguished from non-Hodgkin's lymphomas bythe presence of Reed-Sternberg cells in Hodgkin's lymphomas and theabsence of said cells in non-Hodgkin's lymphomas. Examples ofnon-Hodgkin's lymphomas encompassed by the term as used herein includeany that would be identified as such by one skilled in the art (e.g., anoncologist or pathologist) in accordance with classification schemesknown in the art, such as the Revised European-American Lymphoma (REAL)scheme as described in Color Atlas of Clinical Hematology, ThirdEdition; A. Victor Hofibrand and John E. Pettit (eds.) (HarcourtPublishers Limited 2000) (see, in particular FIG. 11.57, 11.58 and/or11.59). More specific examples include, but are not limited to, relapsedor refractory NHL, front line low grade NHL, Stage III/IV NHL,chemotherapy resistant NHL, precursor B lymphoblastic leukemia and/orlymphoma, small lymphocytic lymphoma, B cell chronic lymphacyticleukemia and/or prolymphocytic leukemia and/or small lymphocyticlymphoma, B-cell prolymphocytic lymphoma, immunocytoma and/orlymphoplasmacytic lymphoma, marginal zone B cell lymphoma, splenicmarginal zone lymphoma, extranodal marginal zone-MALT lymphoma, nodalmarginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasmacell myeloma, low grade/follicular lymphoma, intermediategrade/follicular NHL, mantle cell lymphoma, follicle center lymphoma(follicular), intermediate grade diffuse NHL, diffuse large B-celllymphoma, aggressive NHL (including aggressive front-line NHL andaggressive relapsed NHL), NHL relapsing after or refractory toautologous stem cell transplantation, primary mediastinal large B-celllymphoma, primary effusion lymphoma, high grade immunoblastic NHL, highgrade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulkydisease NHL, Burkitt's lymphoma, precursor (peripheral) T-celllymphoblastic leukemia and/or lymphoma, adult T-cell lymphoma and/orleukemia, T cell chronic lymphocytic leukemia and/or prolymphacyticleukemia, large granular lymphocytic leukemia, mycosis fungoides and/orSezary syndrome, extranodal natural killer/T-cell (nasal type) lymphoma,enteropathy type T-cell lymphoma, hepatosplenic T-cell lymphoma,subcutaneous panniculitis like T-cell lymphoma, skin (cutaneous)lymphomas, anaplastic large cell lymphoma, angiocentric lymphoma,intestinal T cell lymphoma, peripheral T-cell (not otherwise specified)lymphoma and angioimmunoblastic T-cell lymphoma.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer, lungcancer (including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer), pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; multiple myeloma and post-transplant lymphoproliferativedisorder (PTLD). According to one preferred embodiment, the cancercomprises a tumor that expresses a BR3 polypeptide on its surface(BR3-positive). According to another embodiment, the BR3-expressingcancer is a CLL cancer.

In specific embodiments, the anti-BR3 antibodies and polypeptides ofthis invention are used to treat any one or more of the diseasesselected from the group consisting of non-Hodgkin's lymphoma (NHL),lymphocyte predominant Hodgkin's disease (LPHD), chronic lymphocyticleukemia (CLL), acute lymphocytic leukemia (ALL), small lymphocyticlymphoma (SLL), diffuse large B cell lymphoma (DLBCL), follicularlymphoma, which are types of non-Hodgkin's lymphoma (NHL), rheumatoidarthritis and juvenile rheumatoid arthritis, systemic lupuserythematosus (SLE) including lupus nephritis, Wegener's disease,inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP),thrombotic throbocytopenic purpura (TTP), autoimmune thrombocytopenia,multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies,myasthenia gravis, vasculitis, diabetes mellitus, Reynaud's syndrome,Sjorgen's syndrome, glomerulonephritis and multiple myeloma.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², Bi²¹³, P³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.According to one embodiment, the cytotoxic agent is capable of beinginternalized. According to another embodiment, the active portion of thecytotoxic agent is 1100 kD or less. According to one embodiment thechemotherapeutic agent is selected from the group consisting ofmethotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine,etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil,daunorubicin, or other intercalating agents, enzymes and fragmentsthereof such as nucleolytic enzymes, antibiotics, and toxins such assmall molecule toxins or enzymatically active toxins of bacterial,fungal, plant or animal origin, (e.g., monomethylauristatin (MMAE)including fragments and/or variants thereof, and the various antitumoror anticancer agents or grow inhibitory agents disclosed below. Othercytotoxic agents are described below.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlomaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g.,Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; bisphosphonates, such as clodronate; an esperamicin; aswell as neocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), andTAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell in vitro and/or in vivo.Thus, the growth inhibitory agent may be one that significantly reducesthe percentage of cells in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce GI arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), TAXOL® paclitaxel, and topo II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest GI also spill over into S-phase arrest, for example,DNA alkylating agents such as tanoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antieioplastic drugs” by Murakaini et al. (W B Saunders:Philadelphia, 1995), especially p. 13.

An antibody that “induces cell death” is one that causes a viable cellto become nonviable. The cell is generally one that expresses theantigen to which the antibody binds, especially where the celloverexpresses the antigen. Preferably, the cell is a cancer cell, e.g.,a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney,colon, thyroid, pancreatic or bladder cell. In vitro, the cell may be aSKBR3, BT474, Calu 3, MDA-MB-453, MDA-MB-361 or SKOV3 cell. Cell deathin vitro may be determined in the absence of complement and immuneeffector cells to distinguish cell death induced by antibody dependentcell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity(CDC). Thus, the assay for cell death may be performed using heatinactivated serum (i.e. in the absence of complement) and in the absenceof immune effector cells. To determine whether the antibody is able toinduce cell death, loss of membrane integrity as evaluated by uptake ofpropidium iodide (PI), trypan blue (see Moore et al. Cytotechnology,17:1-11 (1995)) or 7AAD can be assessed relative to untreated cells.

An antibody that “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies). Thecell is one which expresses the antigen to which the antibody binds andmay be one that overexpresses the antigen. The cell may be a tumor cell,e.g., a breast, ovarian, stomach, endometrial, salivary gland, lung,kidney, colon, thyroid, pancreatic or bladder cell. In vitro, the cellmay be a SKBR3, BT474, Calu 3 cell, MDA-MB-453, MDA-MB-361 or SKOV3cell. Various methods are available for evaluating the cellular eventsassociated with apoptosis. For example, phosphatidyl serine (PS)translocation can be measured by annexin binding; DNA fragmentation canbe evaluated through DNA laddering as disclosed in the example herein;and nuclear/chromatin condensation along with DNA fragmentation can beevaluated by any increase in hypodiploid cells. Preferably, the antibodythat induces apoptosis is one which results in about 2 to 50 fold,preferably about 5 to 50 fold, and most preferably about 10 to 50 fold,induction of annexin binding relative to untreated cell in an annexinbinding assay using cells expressing the antigen to which the antibodybinds.

Examples of antibodies that induce apoptosis include the anti-DRSantibodies 3F1 1.39.7 (ATCC HB-12456); 3H3.14.5 (ATCC HB-12534);3D5.1.10 (ATCC HB-12536); and 3H3.14.5 (ATCC HB-12534), includinghumanized and/or affinity-matured variants thereof; the human anti-DR5receptor antibodies 16E2 and 20E6, including affinity-matured variantsthereof (WO98/5 1793, expressly incorporated herein by reference); theanti-DR4 antibodies 4E7.24.3 (ATCC HB-12454); 4H6.17.8 (ATCC HB-12455);1H5.25.9 (ATCC HB-12695); 4G7.18.8 (ATCC PTA-99); and 5GI 1.17.1 (ATCCHB-12694), including humanized and/or affinity-matured variants thereof.

In order to screen for antibodies which bind to an epitope on an antigenbound by an antibody of interest, a routine cross-blocking assay such asthat described in Antibodies, A Laboratory Manual, eds. Harlow and Lane(New York: Cold Spring Harbor Laboratory, 1988) can be performed.

A “conjugate” refers to any hybrid molecule, including fusion proteinsand as well as molecules that contain both amino acid or proteinportions and non-protein portions (e.g., toxin-antibody conjugates, orpegylated-antibody conjugates). Conjugates may be synthesized orengineered by a variety of techniques known in the art including, forexample, recombinant DNA techniques, solid phase synthesis, solutionphase synthesis, organic chemical synthetic techniques or a combinationof these techniques. The choice of synthesis will depend upon theparticular molecule to be generated. For example, a hybrid molecule notentirely “protein” in nature may be synthesized by a combination ofrecombinant techniques and solution phase techniques.

According to one embodiment, the conjugate is an antibody or polypeptideof interest covalently linked to a salvage receptor binding epitope(especially an antibody fragment), as described, e.g., in U.S. Pat. No.5,739,277. For example, a nucleic acid molecule encoding the salvagereceptor binding epitope can be linked in frame to a nucleic acidencoding a polypeptide sequence of this invention so that the fusionprotein expressed by the engineered nucleic acid molecule comprises thesalvage receptor binding epitope and a polypeptide sequence of thisinvention. As used herein, the term “salvage receptor binding epitope”refers to an epitope of the Fc region of an IgG molecule (e.g., IgG₁,IgG₂, IgG₃, or IgG₄) that is useful for increasing the in vivo serumhalf-life of the IgG molecule (e.g., Ghetie, V et al., (2000) Ann. Rev.Immunol. 18:739-766, Table 1).

In another embodiment, the conjugate can be formed, by linkage(especially an antibody fragment) to serum albumin or a portion of serumalbumin that binds to the FcRn receptor or a serum albumin-bindingpeptide or to a non-protein polymer (e.g., a polyethylene glycolmoiety). Such polypeptide sequences are disclosed, for example, inWO01/45746. In one preferred embodiment, the serum albumin peptide to beattached comprises an amino acid sequence of DICLPRWGCLW. In anotherembodiment, the half-life of a Fab according to this invention isincreased by these methods. See also, Dennis, M. S., et al., (2002) JBC277(38):35035-35043 for serum albumin binding peptide sequences.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibody.The label may itself be detectable by itself (e.g., radioisotope labelsor fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

A. Compositions and Methods of the Invention

The invention provides antibodies that bind human BR3, and optionallyother primate BR3 as well. According to one embodiment, the H chain hasat least one, two or all of the H chain CDRs of a non-human speciesanti-human BR3 antibody (donor antibody), and substantially all of theframework residues of a human consensus antibody as the recipientantibody. The donor antibody can be from various non-human speciesincluding mouse, rat, guinea pig, goat, rabbit, horse, primate buttypically will be a murine antibody. “Substantially all” in this contextis meant that the recipient FR regions in the humanized antibody mayinclude one or more amino acid substitutions not originally present inthe human consensus FR sequence. These FR changes may comprise residuesnot found in the recipient or the donor antibody.

In one embodiment, the donor antibody is the murine 9.1 antibody, the Vregion including the CDR and FR sequences of each of the VH and VLchains of which are shown in SEQ ID NO:19 and SEQ ID NO:20. In oneembodiment, the residues for the human Fab framework correspond to orwere derived from the consensus sequence of a human Vκ subgroup I and ofa V_(H) subgroup III. According to one embodiment, a humanized BR3antibody of the invention has at least one of the CDRs in the H chain ofthe murine donor antibody. In one embodiment, the humanized BR3 antibodythat binds human BR3 comprises the heavy chain CDRs of the H chain ofthe donor antibody.

In a full length antibody, the humanized BR3 binding antibody of theinvention will comprise a V domain joined to a C domain of a humanimmunoglobulin, e.g., SEQ ID NO:132. In a preferred embodiment, the Hchain C region is from human IgG, such as IgG1 or IgG3. According to oneembodiment, the L chain C domain is from a human κ chain. According toanother embodiment, the Fc sequence of a full length BR3 bindingantibody is SEQ ID NO:134, wherein X is selected from the groupconsisting of N, A, Y, F and H.

The BR3 binding antibodies will bind at least human BR3. According toone embodiment, the BR3-binding antibody will bind other primate BR3such as that of monkeys including cynomolgus and rhesus monkeys, andchimpanzees. According to another embodiment, the BR3 binding antibodyor polypeptide binds a rodent BR3 protein and a human BR3 protein. Inanother embodiment, the BR3 polypeptide binds a mouse BR3 polypeptidesequence and a human BR3 polypeptide sequence.

According to one embodiment, the biological activity of an antagonistBR3 binding antibodies is any one, any combination or all of theactivities selected from the group consisting of (1) binds to a humanBR3 extracellular domain sequence with an apparent Kd value of 500 nM orless, 100 nM or less, 50 nM or less, 10 nM or less, 5 nM or less or 1 nMor less; (2) binds to a human BR3 extracellular domain sequence andbinds to a mouse BR3 extracellular domain sequence with an apparent Kdvalue of 500 nM or less, 100 nM or less, 50nM or less, 10 nM or less, 5nM or less or 1 nM or less; (3) has a functional epitope on humanBR3-comprising residues F25, V33 and A34, wherein the monoclonalantibody; (4) inhibits human BAFF and human BR3 binding; (5) hasantibody dependent cellular cytotoxicity (ADCC) in the presence of humaneffector cells or has increased ADCC in the presence of human effectorcells; (6) binds the human Fc neonatal receptor (FcRn) with a higheraffinity than a polypeptide or parent polypeptide having wild type ornative sequence IgG Fc; (9) kills or depletes B cells in vitro or invivo, preferably by at least 20% when compared to the baseline level orappropriate negative control which is not treated with such antibody;(10) inhibits B cell proliferation in vitro or in vivo and (11) inhibitsB cell survival in vitro or in vivo. According to one embodiment of thepolypeptides or antibodies of this invention, the functional epitopefurther comprises residue R30. According to yet another embodiment ofthis invention, the functional epitope further comprises residues L28and V29.

In one embodiment, compared to treatment with a control antibody thatdoes not bind a B cell surface antigen or as compared to the baselinelevel before treatment, the variable domain of an antibody of thisinvention fused to an Fc region of an mIgG2A can deplete at least 20% ofthe B cells in any one, any combination or all of following populationof cells in mice: (1) B cells in blood, (2) B cells in the lymph nodes,(3) follicular B cells in the spleen and (4) marginal zone B cells inthe spleen. In other embodiments, B cell depletion is 25%, 30%, 40%,50%, 60%, 70%, 80% or greater. In one preferred embodiment, thedepletion is measured at day 15 post treatment with antibody. In anotherpreferred embodiment, the depletion assay is carried out as described inExample 18 or 19 herein. In another preferred embodiment, the depletionis measured by the population of peripheral B cells in a mouse day 15post-treatment.

The desired level of B cell depletion will depend on the disease. Forthe treatment of a BR3 positive cancer, it may be desirable to maximizethe depletion of the B cells which are the target of the anti-BR3antibodies and polypeptides of the invention. Thus, for the treatment ofa BR3 positive B cell neoplasm, it is desirable that the B celldepletion be sufficient to at least prevent progression of the diseasewhich can be assessed by the physician of skill in the art, e.g., bymonitoring tumor growth (size), proliferation of the cancerous celltype, metastasis, other signs and symptoms of the particular cancer.According to one preferred embodiment, the B cell depletion issufficient to prevent progression of disease for at least 2 months, morepreferably 3 months, even more preferably 4 months, more preferably 5months, even more preferably 6 or more months. In even more preferredembodiments, the B cell depletion is sufficient to increase the time inremission by at least 6 months, more preferably 9 months, morepreferably one year, more preferably 2 years, more preferably 3 years,even more preferably 5 or more years. In a most preferred embodiment,the B cell depletion is sufficient to cure the disease. In preferredembodiments, the B cell depletion in a cancer patient is at least about75% and more preferably, 80%, 85%, 90%, 95%, 99% and even 100% of thebaseline level before treatment.

For treatment of an autoimmune disease, it can be desirable to modulatethe extent of B cell depletion depending on the disease and/or theseverity of the condition in the individual patient, by adjusting thedosage of BR3 binding antibody or polypeptide. Thus, B cell depletioncan but does not have to be complete. Total B cell depletion may bedesired in initial treatment but in subsequent treatments, the dosagemay be adjusted to achieve only partial depletion. In one embodiment,the B cell depletion is at least 20%, i.e., 80% or less of BR3 positiveB cells remain as compared to the baseline level before treatment. Inother embodiments, B cell depletion is 25%, 30%, 40%, 50%, 60%, 70%, 80%or greater. According to one preferred embodiment, the B cell depletionis sufficient to halt progression of the disease, more preferably toalleviate the signs and symptoms of the particular disease undertreatment, even more preferably to cure the disease.

The invention also provides bispecific BR3 binding antibodies whereinone arm of the antibody has a humanized H and L chain of the BR3 bindingantibody of the invention, and the other arm has V region bindingspecificity for a second antigen. In specific embodiments, the secondantigen is selected from the group consisting of CD3, CD64, CD32A, CD16,NKG2D or other NK activating ligands.

Any cysteine residue not involved in maintaining the proper conformationof the anti-BR3 antibody also may be substituted, generally with serine,to improve the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g. 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and human BR3. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of theanti-BR3 antibody are prepared by a variety of methods known in the art.These methods include, but are not limited to, isolation from a naturalsource (in the case of naturally occurring amino acid sequence variants)or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the anti-BR3 antibody.

It may be desirable to modify the antibody of the invention with respectto effector function, e.g. so as to enhance antigen-dependentcell-mediated cyotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al. Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement mediated lysis and ADCCcapabilities. See Stevenson et al. Anti-Cancer Drug Design 3:219-230(1989).

To increase the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

Other Antibody Modifications

Other modifications of the antibody are contemplated herein. Forexample, the antibody may be linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, polyoxyalkylenes, or copolymers of polyethylene glycol andpolypropylene glycol. The antibody also may be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,(1980).

Screening for Antibodies with the Desired Properties

Antibodies with certain biological characteristics may be selected asdescribed in the Experimental Examples. For example, antibodies thatbind BR3 can be selected by binding to BR3 in ELISA assays or, morepreferably, by binding to BR3 expressed on the surface of cells (e.g.,BJAB cell line). See, e.g., Example 2.

The growth inhibitory effects of an anti-BR3 antibody of the inventionmay be assessed by the Examples or methods known in the art, e.g., usingcells which express BR3 either endogenously or following transfectionwith the BR3 gene. For example, in one preferred embodiment, primary Bcells expressing BR3 can be used in proliferation and survival assays(e.g., Example 7). In another example, tumor cell lines andBR3-transfected cells may treated with an anti-BR3 monoclonal antibodyof the invention at various concentrations for a few days (e.g., 2-7)days and stained with crystal violet or MTT or analyzed by some othercolorimetric assay. Another method of measuring proliferation would beby comparing ³H-thymidine uptake by the cells treated in the presence orabsence an anti-BR3 antibody of the invention. After antibody treatment,the cells are harvested and the amount of radioactivity incorporatedinto the DNA quantitated in a scintillation counter. Appropriatepositive controls include treatment of a selected cell line with agrowth inhibitory antibody known to inhibit growth of that cell line.

To select for antibodies which induce cell death, loss of membraneintegrity as indicated by, e.g., propidium iodide (PI), trypan blue or7AAD uptake may be assessed relative to control. A PI uptake assay canbe performed in the absence of complement and immune effector cells.BR3-expressing tumor cells are incubated with medium alone or mediumcontaining of the appropriate monoclonal antibody at e.g, about 10μg/ml. The cells are incubated for a 3 day time period. Following eachtreatment, cells are washed and aliquoted into 35 mm strainer-capped12×75 tubes (1 ml per tube, 3 tubes per treatment group) for removal ofcell clumps. Tubes then receive PI (10 μg/ml). Samples may be analyzedusing a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software(Becton Dickinson). Those antibodies which induce statisticallysignificant levels of cell death as determined by PI uptake may beselected as cell death-inducing antibodies.

To screen for antibodies which bind to an epitope on BR3 bound by anantibody of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed. Thisassay can be used to determine if a test antibody binds the same site orepitope as an anti-BR3 antibody of the invention. Alternatively, oradditionally, epitope mapping can be performed by methods known in theart. For example, the antibody sequence can be mutagenized such as byalanine scanning, to identify contact residues. The mutant antibody isinitailly tested for binding with polyclonal antibody to ensure properfolding. In a different method, peptides corresponding to differentregions of BR3 can be used in competition assays with the testantibodies or with a test antibody and an antibody with a characterizedor known epitope.

Examples of Specific Anti-BR3Antibodies

Antibodies of this invention specifically include antibodies comprisingthe variable heavy chain sequence of any one of the antibodies disclosedin Table 2 (below), and BR3-binding fragments thereof that has not beenproduced by a hybridoma cell. Antibodies of this invention specificallyinclude antibodies comprising a variable heavy chain sequence comprisingthe sequence of any one of SEQ ID NO: 4-13, 15-18, 22, 24, 26-73, 75-76,78, 80-85, 87-96, 98, 100, 102, 104, 106-107, 109-110, 112, 114, 116,118, 120, 122, 124, 126 and 127, and BR3-binding fragments thereof.According to a further embodiment, an antibody of this inventioncomprises the variable heavy and the variable light chain region of anyone of the antibodies disclosed in Table 2, and BR3-binding fragmentsthereof. According to one embodiment, the antibody further comprises anFc region comprising the sequence of SEQ ID NO:134, wherein X is anamino acid selected from the group consisting of N, A, W, Y, F and H.According to another embodiment, the antibody comprises the sequence ofSEQ ID NO:76 or SEQ 131 NO:131, wherein X is an amino acid selected fromthe group consisting of N, A, W, Y, F and H.

TABLE 2 Examples of Antibody Sequences SEQ ID SEQ ID ANTIBODY NO: NO:FRAMEWORK 2.1   1 (VL)   2 (VH) Mouse hu2.1-Graft   3 (VL)   4 (VH)R71A/N73T/L78A Hu2.1-RL   3 (VL)   5 (VH) RL Hu2.1-RE   3 (VL)   6 (VH)RF Hu2.1-40   3 (VL)   7 (VH) RF Hu2.1-46   3 (VL)   8 (VH) RF Hu2.1-30  3 (VL)   9 (VH) RF Hu2.1-93   3 (VL)  10 (VH) RL Hu2.1-94   3 (VL)  11(VH) RL Hu2.1-40L   3 (VL)  12 (VH) RL Hu2.1-89   3 (VL)  13 (VH) RLHu2.1-46.DANA-  14 (LC)  15 (HC) RF IgG Hu2.1-27   3 (VL)  16 (VH) RFHu2.1-36   3 (VL)  17 (VH) RF Hu2.1-31   3 (VL)  18 (VH) RF 9.1  19 (VL) 20 (VH) Mouse Hu9.1-graft  21 (VL)  22 (VH) R71A/N73T/L78A Hu9.1-73  23(VL)  24 (VH) R71A/N73T/L78A Hu9.1-70  25 (VL)  26 (VH) R71A/N73T/L78AHu9.1-56  21 (VL)  27 (VH) R71A/N73T/L78A Hu9.1-51  21 (VL)  28 (VH)R71A/N73T/L78A Hu9.1-59  21 (VL)  29 (VH) R71A/N73T/L78A Hu9.1-61  21(VL)  30 (VH) R71A/N73T/L78A Hu9.1-A  21 (VL)  31 (VH) R71A/N73T/L78AHu9.1-B  21 (VL)  32 (VH) R71A/N73T/L78A Hu9.1-C  21 (VL)  33 (VH)R71A/N73T/L78A Hu9.1-66  21 (VL)  34 (VH) R71A/N73T/L78A Hu9.1-RF  21(VL)  35 (VH) RF Hu9.1-48  21 (VL)  36 (VH) RF Hu9.1-RL  21 (VL)  37(VH) RL Hu9.1-91  21 (VL)  38 (VH) RL Hu9A-90  21 (VL)  39 (VH) RLHu9.1-75  21 (VL)  40 (VH) RL Hu9.1-88  21 (VL)  41 (VH) RL Hu9.1RL-9 21 (VL)  42 (VH) RL Hu9.1RL-44  21 (VL)  43 (VH) RL Hu9.1RL-13  21 (VL) 44 (VH) RL Hu9.1RL-47  21 (VL)  45 (VH) RL Hu9.1RL-28  21 (VL)  46 (VH)RL Hu9.1RL-43  21 (VL)  47 (VH) RL Hu9.1RL-16  21 (VL)  48 (VH) RLHu9.1RL-70  21 (VL)  49 (VH) RL Hu9.1RL-30  21 (VL)  50 (VH) RLHu9.1RL-32  21 (VL)  51 (VH) RL Hu9.1RL-37  21 (VL)  52 (VH) RLHu9.1RL-29  21 (VL)  53 (VH) RL Hu9.1RL-10  21 (VL)  54 (VH) RLHu9.1RL-24  21 (VL)  55 (VH) RL Hu9.1RL-39  21 (VL)  56 (VH) RLHu9.1RL-31  21 (VL)  57 (VH) RL Hu9.1RL-18  21 (VL)  58 (VH) RLHu9.1RL-23  21 (VL)  59 (VH) RL Hu9.1RL-41  21 (VL)  60 (VH) RLHu9.1RL-95  21 (VL)  61 (VH) RL Hu9.1RL-14  21 (VL)  62 (VH) RLHu9.1RL-57  21 (VL)  63 (VH) RL Hu9.1RL-15  21 (VL)  64 (VH) RLHu9.1RL-54  21 (VL)  65 (VH) RL Hu9.1RL-12  21 (VL)  66 (VH) RLHu9.1RL-34  21 (VL)  67 (VH) RL Hu9.1RL-25  21 (VL)  68 (VH) RLHu9.1RL-71  21 (VL)  69 (VH) RL Hu9.1RL-5  21 (VL)  70 (VH) RLHu9.1RL-79  21 (VL)  71 (VH) RL Hu9.1RL-66  21 (VL)  72 (VH) RLHu9.1RL-69  21 (VL)  73 (VH) RL 9.1RF-IgG  74 (LC)  75 (HC) RF 9.1RF-IgG 74 (LC)  76 (HC) RF (N434X) 11G9  77 (VL)  78 (VH) Mouse Hu11G9-graft 79 (VL)  80 (VH) R71A/N73T/L78A Hu11G9-RF  79 (VL)  81 (VH) RFHu11G9-36  79 (VL)  82 (VH) RF Hu11G9-46  79 (VL)  83 (VH) RF Hu11G9-35 79 (VL)  84 (VH) RF Hu11G9-29  79 (VL)  85 (VH) RF V3-Fab  86 (LC)  87(HC) V24  86 (VL)  88 (VH) V44  86 (VL)  89 (VH) V89  86 (VL)  90 (VH)V96  86 (VL)  91 (VH) V46  86 (VL)  92 (VH) V51  86 (VL)  93 (VH) V75 86 (VL)  94 (VH) V58  86 (VL)  95 (VH) V60  86 (VL)  96 (VH) V3-1  97(VL)  98 (VH) V3-11  99 (VL) 100 (VH) V3-12 101 (VL) 102 (VH) V3-13 103(VL) 104 (VH) V3-3 105 (VL) 106 (VH) V3-5  97 (VL) 107 (VH) V3-9 108(VL)  98 (VH) V3-16  97 (VL) 109 (VH) V3-19  97 (VL) 110 (VH) V3-24 111(VL) 112 (VH) V3-27 113 (VL) 114 (VH) V3-34 115 (VL) 116 (VH) V3-35 117(VL) 118 (VH) V3-37 119 (VL) 120 (VH) V3-41 121 (VL) 122 (VH) V3-46 123(VL) 124 (VH) V3-46a 123 (VL) 125 (VH) V3-46q 123 (VL) 126 (VH) V3-46s123 (VL) 127 (VH) V3-46sFab 128 (LC) 129 (VH) V3-46s IgG 128 (LC) 130(VH) V3-46s IgG 128 (LC) 131 (VH) (N434X) V3-46s-1 194 (LC) 127 (VH)V3-46s-7 195 (LC) 127 (VH) V3-46s-9 196 (LC) 127 (VH) V3-46s-10 197 (LC)127 (VH) V3-46s-12 198 (LC) 193 (VH) V3-46s-13 199 (LC) 127 (VH)V3-46s-29 200 (LC) 127 (VH) V3-46s-31 201 (LC) 127 (VH) V3-46s-33 202(LC) 127 (VH) V3-46s-34 203 (LC) 127 (VH) V3-46s-37 204 (LC) 127 (VH)V3-46s-40 205 (LC) 127 (VH) V3-46s-42 206 (LC) 127 (VH) V3-46s-45 207(LC) 127 (VH)

Antibodies of this invention include BR3-binding antibodies having an H3sequence that is at least about 70% amino acid sequence identity,alternatively at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity,to the H3 sequence of any one of the sequences of SEQ ID NO:s: 4-13,15-18, 22, 24, 26-73, 75-76, 78, 80-85, 87-96, 98, 100, 102, 104,106-107, 109-110, 112, 114, 116, 118, 120, 122, 124, 126 and 127, andBR3 binding fragments of those antibodies.

Antibodies of this invention include BR3-binding antibodies having H1,H2 and H3 sequences that are at least 70% identical to the underlinedportions of any one of the antibodies sequences described in the Figuresor to the CDRs of hypervariable regions described in the SequenceListing, or alternatively at least about 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequenceidentical.

Antibodies of this invention include BR3-binding antibodies having L1,L2 and L3 sequences that are at least 70% identical to the underlinedportions of any one of the antibodies sequences described in the Figuresor to the CDRs or hypervariable regions described in the SequenceListing, or alternatively at least about 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequenceidentical.

Antibodies of this invention include BR3-binding antibodies having a VHdomain with at least 70% homology to a VH domain of any one of theantibodies of Table 2, or alternatively at least about 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% aminoacid sequence identical.

Antibodies of this invention include any BR3-binding antibody comprisinga heavy chain CDR3 sequence of an antibody sequence of Table 2 that hasnot been produced by a hybridoma cell. Antibodies of this inventioninclude any BR3-binding antibody comprising a heavy chain CDR3 sequenceof any one of SEQ ID NO:s:7-13, 15-18, 36, 38-73, 78, 82-85, 87-96, 98,100, 102, 104, 106-107, 109-110, 112, 114, 116, 118, 120, 122, 124, 126and 127, or comprising a H3 sequence that is derived a H3 sequence ofany one of SEQ ID NO:s:7-13, 15-18, 36, 38-73, 78, 82-85, 87-96, 98,100, 102, 104, 106-107, 109-110, 112, 114, 116, 118, 120, 122, 124, 126and 127. In another embodiment, an antibody of this invention includesany BR3-binding antibody comprising a CDR-H1, CDR-H2 and CDR-H3 of anyone of the sequences selected from the group consisting of SEQ IDNOs:7-13, 15-18, 36, 38-73, 78, 82-85, 87-96, 98, 100, 102, 104,106-107, 109-110, 112, 114, 116, 118, 120, 122, 124, 126 and 127 or isderived from an antibody comprising the CDR-H1, CDR-H2 and CDR-H3sequences. Antibodies of this invention include any BR3-binding antibodycomprising a heavy chain H1, H2 and 1-13 sequence of an antibody ofTable 2 that has not been produced by a hybridoma cell.

Antibodies of this invention include the antibodies comprising apolypeptide sequence encoded by the Hu9.1-RF-H-IgG nucleic acid sequencedeposited as ATCC deposit number PTA-6315 on Nov. 17, 2004 and anti-BR3binding antibodies that comprise an amino acid sequence that is at least70% identical, alternatively at least about 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acidsequence identical, to any one of the variable regions sequence of theHu9.1-RF-H-IgG polypeptide sequence. Antibodies of this inventioninclude the antibodies comprising a polypeptide sequence encoded by theHu9.1-RF-L-IgG nucleic acid sequence deposited as ATCC deposit numberPTA-6316 on Nov. 17, 2004 and anti-BR3 binding antibodies that comprisean amino acid sequence that is at least 70% identical, alternatively atleast about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% amino acid sequence identical, to the variableregion sequence of the Hu9.1-RF-L-IgG polypeptide sequence.

Antibodies of this invention include the antibodies comprising apolypeptide sequence encoded by the Hu2.1-46. DANA-H-IgG nucleic acidsequence deposited as ATCC deposit number PTA-6313 on Nov. 17, 2004 andanti-BR3 binding antibodies that comprise an amino acid sequence that isat least 70% identical, alternatively at least about 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acidsequence identical, to the variable region sequence of the Hu2.1-46.DANA-H-IgG polypeptide sequence. Antibodies of this invention includethe antibodies comprising a polypeptide sequence encoded by theHu2.1-46. DANA-L-IgG nucleic acid sequence deposited as ATCC depositnumber PTA-6314 on Nov. 17, 2004 and anti-BR3 binding antibodies thatcomprise an amino acid sequence that is at least 70% identical,alternatively at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identical,to the variable region sequence of the Hu2.1-46. DANA-L-IgG polypeptidesequence.

Antibodies of this invention include the antibodies comprising apolypeptide sequence encoded by the HuV3-46s-H-IgG nucleic acid sequencedeposited as ATCC deposit number PTA-6317 on Nov. 17, 2004 and anti-BR3binding antibodies that comprise an amino acid sequence that is at least70% identical, alternatively at least about 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%; 84%, 85%, 86%, 87%, 88%, 89%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acidsequence identical, to the variable region sequence of theHuV3-46s-H-IgG polypeptide sequence. Antibodies of this inventioninclude the antibodies comprising a polypeptide sequence encoded by theHuV3-46s-L-IgG nucleic acid sequence deposited as ATCC deposit numberPTA-6318 on Nov. 17, 2004 and anti-BR3 binding antibodies that comprisean amino acid sequence that is at least 70% identical, alternatively atleast about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% amino acid sequence identical, to the variableregion sequence of the HuV3-46s-L-IgG polypeptide sequence.

Antibodies of this invention include the Hu9.1-RF-IgG antibodycomprising the heavy chain sequence of ATCC deposit no. PTA-6315 and thelight chain sequence of ATCC deposit no. PTA-6316. Antibodies of thisinvention include the Hu2.1-46. DANA-IgG antibody comprising the heavysequence of ATCC deposit no. PTA-6313 and the light chain sequence ofATCC deposit no. PTA-6314. Antibodies of this invention include theHuV3-46s-IgG antibody comprising the heavy sequence of ATCC deposit no.PTA-6317 and the light chain sequence of ATCC deposit no. PTA-6318.

According to one preferred embodiment, the antibodies of this inventionspecifically bind to a sequence of a native human BR3 polypeptide.According to yet another embodiment, an antibody of this invention hasimproved binding to the FcRn receptor at pH 6.0 compared to the antibodyknown as 9.1-RF Ig. According to yet another embodiment, an antibody ofthis invention has improved ADCC function in the presence of humaneffector cells compared to the antibody known as 9.1-RF Ig. According toyet another embodiment, an antibody of this invention has decreased ADCCfunction in the presence of human effector cells compared to theantibody known as 9.1-RF Ig.

It is understood that all antibodies of this invention includeantibodies lacking a signal sequence and antibodies lacking the K447residue of the Fc region.

Vectors, Host Cells and Recombinant Methods

The invention also provides an isolated nucleic acid encoding a BR3binding antibody or BR3 binding polypeptide, vectors and host cellscomprising the nucleic acid, and recombinant techniques for theproduction of the antibody.

For recombinant production of the BR3 binding antibodies andpolypeptides, the nucleic acid encoding it is isolated and inserted intoa replicable vector for further cloning (amplification of the DNA) orfor expression. DNA encoding the monoclonal antibody or polypeptide isreadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of the antibody). Many vectorsare available. The vector components generally include, but are notlimited to, one or more of the following: a signal sequence, an originof replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

(i) Signal Sequence Component

The antibody or polypeptide of this invention may be producedrecombinantly not only directly, but also as a fusion polypeptide with aheterologous polypeptide, which is preferably a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature protein or polypeptide. The heterologous signal sequence selectedpreferably is one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. For prokaryotic host cells that donot recognize and process the native BR3 binding antibody signalsequence, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group of the alkalinephosphatase, penicillinase, 1 pp, or heat-stable enterotoxin II leaders.For yeast secretion the native signal sequence may be substituted by,e.g., the yeast invertase leader, a factor leader (includingSaccharomyces and Kluyveromyces α-factor leaders), or acid phosphataseleader, the C. albicans glucoamylase leader, or the signal described inWO 90/13646. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the BR3 binding antibody.

(ii) Origin of Replication

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theBR3 binding antibody nucleic acid, such as DHFR, thymidine kinase,metallothionein-I and -II, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mix), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCCCRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding BR3 binding antibody, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the BR3 binding antibody. Promoters suitable for use withprokaryotic hosts include the phoA promoter, β-lactamase and lactosepromoter systems, alkaline phosphatase promoter, a tryptophan (trp)promoter system, and hybrid promoters such as the tac promoter. However,other known bacterial promoters are suitable. Promoters for use inbacterial systems also will contain a Shine-Dalgarno (S.D.) sequenceoperably linked to the DNA encoding the BR3 binding antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoter sequences for use with yeast hosts includethe promoters for 3-phosphoglycerate kinase or other glycolytic enzymes,such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells can becontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40(SV40), or from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding an antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270); the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elementsfor activation of eukaryotic promoters. The enhancer may be spliced intothe vector at a position 5′ or 3′ to the antibody-encoding sequence, butis preferably located at a site 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.

(vii) Selection and transformation of host cells Suitable host cells forcloning or expressing the DNA in the vectors herein are the prokaryote,yeast, or higher eukaryote cells described above. Suitable prokaryotesfor this purpose include eubacteria, such as Gram-negative orGram-positive organisms, for example, Enterobacteriaceae such asEscherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratiamarcescans, and Shigella, as well as Bacilli such as B. subtilis and B.licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, andStreptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X1776 (ATCC31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examplesare illustrative rather than limiting.

Full length antibody, antibody fragments, and antibody fusion proteinscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) and theimmunoconjugate by itself shows effectiveness in tumor cell destruction.Full length antibodies have greater half life in circulation. Productionin E. coli is faster and more cost efficient. For expression of antibodyfragments and polypeptides in bacteria, see, e.g., U.S. Pat. No.5,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), andU.S. Pat. No. 5,840,523 (Simmons et al.) which describes translationinitiation region (TIR) and signal sequences for optimizing expressionand secretion, these patents incorporated herein by reference. Afterexpression, the antibody is isolated from the E. coli cell paste in asoluble fraction and can be purified through, e.g., a protein A or Gcolumn depending on the isotype. Final purification can be carried outsimilar to the process for purifying antibody expressed e.g., in CHOcells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for BR3 bindingantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated BR3 bindingantibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(ix) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, hydrophobic interactionchromatography, gel electrophoresis, dialysis, and affinitychromatography, with affinity chromatography being among one of thetypically preferred purification steps. The suitability of protein A asan affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Antibody Conjugates

The antibody may be conjugated to a cytotoxic agent, such as achemotherapeutic agent, toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate). In certain embodiments,the toxin is calicheamicin, a maytansinoid, a dolastatin, auristatin Eand analogs or derivatives thereof, are preferable.

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described herein. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

Preferred drugs/toxins include DNA damaging agents, inhibitors ofmicrotubule polymerization or depolymerization and antimetabolites.Preferred classes of cytotoxic agents include, for example, the enzymeinhibitors such as dihydrofolate reductase inhibitors, and thymidylatesynthase inhibitors, DNA intercalators, DNA cleavers, topoisomeraseinhibitors, the anthracycline family of drugs, the vinca drugs, themitomycins, the bleomycins, the cytotoxic nucleosides, the pteridinefamily of drugs, diynenes, the podophyllotoxins and differentiationinducers. Particularly useful members of those classes include, forexample, methotrexate, methopterin, dichloromethotrexate,5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan,leurosine, leurosideine, actinomycin, daunorubicin, doxorubicin,N-(5,5-diacetoxypentyl)doxorubicin, morpholino-doxorubicin,1-(2-choroehthyl)-1,2-dimethanesulfonyl hydrazide, N⁸-acetyl spermidine,aminopterin methopterin, esperamicin, mitomycin C, mitomycin A,actinomycin, bleomycin, caminomycin, aminopterin, tallysomycin,podophyllotoxin and podophyllotoxin derivatives such as etoposide oretoposide phosphate, vinblastine, vincristine, vindesine, taxol,taxotere, retinoic acid, butyric acid, N⁸⁻acetyl spermidine,camptothecin, calicheamicin, bryostatins, cephalostatins, ansamitocin,actosin, maytansinoids such as DM-1, maytansine, maytansinol,N-desmethyl-4,5-desepoxymaytansinol, C-19-dechloromaytansinol,C-20-hydroxymaytansinol, C-20-demethoxymaytansinol, C-9-SH maytansinol,C-14-alkoxymethylmaytansinol, C-14-hydroxy or acetyloxymethlmaytansinol,C-15-hydroxy/acetyloxymaytansinol, C-15-methoxymaytansinol,C-18-N-demethylmaytansinol and 4,5-deoxymaytansinol, auristatins such asauristatin E, M, PHE and PE; dolostatins such as dolostatin A,dolostatin B, dolostatin C, dolostatin D, dolostatin E (20-epi and11-epi), dolostatin G, dolostatin H, dolostatin I, dolostatin 1,dolostatin 2, dolostatin 3, dolostatin 4, dolostatin 5, dolostatin 6,dolostatin 7, dolostatin 8, dolostatin 9, dolostatin 10, deo-dolostatin10, dolostatin 11, dolostatin 12, dolostatin 13, dolostatin 14,dolostatin 15, dolostatin 16, dolostatin 17, and dolostatin 18;cephalostatins such as cephalostatin 1, cephalostatin 2, cephalostatin3, cephalostatin 4, cephalostatin 5, cephalostatin 6, cephalostatin 7,25′-epi-cephalostatin 7,20-epi-cephalostatin 7, cephalostatin 8,cephalostatin 9, cephalostatin 10, cephalostatin 11, cephalostatin 12,cephalostatin 13, cephalostatin 14, cephalostatin 15, cephalostatin 16,cephalostatin 17, cephalostatin 18, and cephalostatin 19.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533, the disclosures of which are hereby expressly incorporated byreference.

Maytansine and maytansinoids have been conjugated to antibodiesspecifically binding to tumor cell antigens. Immunoconjugates containingmaytansinoids and their therapeutic use are disclosed, for example, inU.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1,the disclosures of which are hereby expressly incorporated by reference.Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describedimmunoconjugates comprising a maytansinoid designated DM1 linked to themonoclonal antibody C242 directed against human colorectal cancer. Theconjugate was found to be highly cytotoxic towards cultured colon cancercells, and showed antitumor activity in an in vivo tumor growth assay.Chari et al. Cancer Research 52:127-131 (1992) describe immunoconjugatesin which a maytansinoid was conjugated via a disulfide linker to themurine antibody A7 binding to an antigen on human colon cancer celllines, or to another murine monoclonal antibody TA.1 that binds theHER-2/neu oncogene.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, andChari et at Cancer Research 52: 127-131 (1992). The linking groupsinclude disufide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups, or esterase labile groups,as disclosed in the above-identified patents, disulfide and thioethergroups being preferred.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 [1978]) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhyrdoxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

Calicheamicin

Another immunoconjugate of interest comprises an BR3 binding antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. For the preparation ofconjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001,5,877,296 (all to American Cyanamid Company). Structural analogues ofcalicheamicin which may be used include, but are not limited to, γ₁^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ¹ ₁ (Hinman et al.Cancer Research 53: 3336-3342 (1993), Lode et al. Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Radioactive Isotopes

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated anti-BR3 antibodies. Examplesinclude At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹²and radioactive isotopes of Lu. When the conjugate is used fordiagnosis, it may comprise a radioactive atom for scintigraphic studies,for example tc^(99m) or I¹²³, or a spin label for nuclear magneticresonance (NMR) imaging (also known as magnetic resonance imaging, mri),such as iodine-123 again, iodine-131, indium-111, fluorine-19,carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al. Cancer Research 52: 127-131(1992); U.S. Pat. No. 5,208,020) may be used.

In another embodiment, the antibody can be conjugated to a “receptor”(such as streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is conjugatedto a cytotoxic agent (e.g., a radionucleotide).

Therapeutic Uses of the BR3 Binding Antibodies

The BR3 binding antibodies of the invention are useful to treat a numberof malignant and non-malignant diseases including autoimmune diseasesand related conditions, and cancers, including BR3 positive cancersincluding B cell lymphomas and leukemias. Stem cells (B-cellprogenitors) in bone marrow lack the BR3 antigen, allowing healthyB-cells to regenerate after treatment and return to normal levels withinseveral months.

Autoimmune diseases or autoimmune related conditions include arthritis(rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis,psoriatic arthritis), psoriasis, dermatitis including atopic dermatitis;chronic autoimmune urticaria, polymyositis/dermatomyositis, toxicepidermal necrolysis, systemic scleroderma and sclerosis, responsesassociated with inflammatory bowel disease (IBD) (Crohn's disease,ulcerative colitis), respiratory distress syndrome, adult respiratorydistress syndrome (ARDS), meningitis, allergic rhinitis, encephalitis,uveitis, colitis, glomerulonephritis, allergic conditions, eczema,asthma, conditions involving infiltration of T cells and chronicinflammatory responses, atherosclerosis, autoimmune myocarditis,leukocyte adhesion deficiency, systemic lupus erythematosus (SLE), lupus(including nephritis, non-renal, discoid, alopecia), juvenile onsetdiabetes, multiple sclerosis, allergic encephalomyelitis, immuneresponses associated with acute and delayed hypersensitivity mediated bycytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosisincluding Wegener's granulomatosis, agranulocytosis, vasculitis(including ANCA), aplastic anemia, Coombs positive anemia, DiamondBlackfan anemia, immune hemolytic anemia including autoimmune hemolyticanemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), FactorVIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia,leukopenia, diseases involving leukocyte diapedesis, CNS inflammatorydisorders, multiple organ injury syndrome, myasthenia gravis,antigen-antibody complex mediated diseases, anti-glomerular basementmembrane disease, anti-phospholipid antibody syndrome, allergicneuritis, Bechet disease, Castleman's syndrome, Goodpasture's Syndrome,Lambert-Eaton Myasthenic Syndrome, Reynaud's syndrome, Sjorgen'ssyndrome, Stevens-Johnson syndrome, solid organ transplant rejection(including pretreatment for high panel reactive antibody titers, IgAdeposit in tissues, etc), graft versus host disease (GVHD), pemphigoidbullous, pemphigus (all including vulgaris, foliatis), autoimmunepolyendocrinopathies, Reiter's disease, stiff-man syndrome, giant cellarteritis, immune complex nephritis, IgA nephropathy, IgMpolyneuropathies or IgM mediated neuropathy, idiopathic thrombocytopenicpurpura (ITP), thrombotic throbocytopenic purpura (TTP), autoimmunethrombocytopenia, autoimmune disease of the testis and ovary includingautoimune orchitis and oophoritis, primary hypothyroidism; autoimmuneendocrine diseases including autoimmune thyroiditis, chronic thyroiditis(Hashimoto's Thyroiditis), subacute thyroiditis, idiopathichypothyroidism, Addison's disease, Grave's disease, autoimmunepolyglandular syndromes (or polyglandular endocrinopathy syndromes),Type I diabetes also referred to as insulin-dependent diabetes mellitus(IDDM) and Sheehan's syndrome; autoimmune hepatitis, Lymphoidinterstitial pneumonitis (HIV), bronchiolitis obliterans(non-transplant) vs NSIP, Guillain-Barre' Syndrome, Large VesselVasculitis (including Polymyalgia Rheumatica and Giant Cell (Takayasu's)Arteritis), Medium Vessel Vasculitis (including Kawasaki's Disease andPolyarteritis Nodosa), ankylosing spondylitis, Berger's Disease (IgAnephropathy), Rapidly Progressive Glomerulonephritis, Primary biliarycirrhosis, Celiac sprue (gluten enteropathy), Cryoglobulinemia, ALS,coronary artery disease.

BR3 positive cancers are those comprising abnormal proliferation ofcells that express BR3 on the cell surface. The BR3 positive B cellneoplasms include BR3-positive Hodgkin's disease including lymphocytepredominant Hodgkin's disease (LPHD); non-Hodgkin's lymphoma (NHL);follicular center cell (FCC) lymphomas; acute lymphocytic leukemia(ALL); chronic lymphocytic leukemia (CLL); Hairy cell leukemia. Thenon-Hodgkins lymphoma include low grade/follicular non-Hodgkin'slymphoma (NHL), small lymphocytic lymphoma (SLL), intermediategrade/follicular NHL, intermediate grade diffuse NHL, high gradeimmunoblastic NHL, high grade lymphoblastic NHL, high grade smallnon-cleaved cell NHL, bulky disease NHL, plasmacytoid lymphocyticlymphoma, mantle cell lymphoma, AIDS—related lymphoma and Waldenstrom'smacroglobulinemia. Treatment of relapses of these cancers are alsocontemplated. LPHD is a type of Hodgkin's disease that tends to relapsefrequently despite radiation or chemotherapy treatment and ischaracterized by BR3-positive malignant cells. CLL is one of four majortypes of leukemia. A cancer of mature B-cells called lymphocytes, CLL ismanifested by progressive accumulation of cells in blood, bone marrowand lymphatic tissues.

In specific embodiments, the BR3 binding antibodies and functionalfragments thereof are used to treat non-Hodgkin's lymphoma (NHL),lymphocyte predominant Hodgkin's disease (LPHD), small lymphocyticlymphoma (SLL), chronic lymphocytic leukemia, rheumatoid arthritis andjuvenile rheumatoid arthritis, systemic lupus erythematosus (SLE)including lupus nephritis, Wegener's disease, inflammatory boweldisease, idiopathic thrombocytopenic purpura (ITP), thromboticthrobocytopenic purpura (TTP), autoimmune thrombocytopenia, multiplesclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, myastheniagravis, vasculitis, diabetes mellitus, Reynaud's syndrome, Sjorgen'ssyndrome and glomerulonephritis.

The BR3 binding antibodies or functional fragments thereof are useful asa single-agent treatment in, e.g., for relapsed or refractory low-gradeor follicular, BR3-positive, B-cell NHL, or can be administered topatients in conjunction with other drugs in a multi drug regimen.

Indolent lymphoma is a slow-growing, incurable disease in which theaverage patient survives between six and 10 years following numerousperiods of remission and relapse. In one embodiment, the humanized BR3binding antibodies or functional fragments thereof are used to treatindolent NHL.

The parameters for assessing efficacy or success of treatment of theneoplasm will be known to the physician of skill in the appropriatedisease. Generally, the physician of skill will look for reduction inthe signs and symptoms of the specific disease. Parameters can includemedian time to disease progression, time in remission, stable disease.

The following references describe lymphomas and CLL, their diagnoses,treatment and standard medical procedures for measuring treatmentefficacy.

The following references describe lymphomas and CLL, their diagnoses,treatment and standard medical procedures for measuring treatmentefficacy. Canellos G P, Lister, T A, Sklar J L: The Lymphomas. W.B.Saunders Company, Philadelphia, 1998; van Besien K and Cabanillas, F:Clinical Manifestations, Staging and Treatment of Non-Hodgkin'sLymphoma, Chap. 70, pp 1293-1338, in: Hematology, Basic Principles andPractice, 3rd ed. Hoffman et al. (editors). Churchill Livingstone,Philadelphia, 2000; and Rai, K and Patel, D:Chronic LymphocyticLeukemia, Chap. 72, pp 1350-1362; in: Hematology, Basic Principles andPractice, 3rd ed. Hoffman et al. (editors). Churchill Livingstone,Philadelphia, 2000.

The parameters for assessing efficacy or success of treatment of anautoimmune or autoimmune related disease will be known to the physicianof skill in the appropriate disease. Generally, the physician of skillwill look for reduction in the signs and symptoms of the specificdisease. The following are by way of examples.

In one embodiment, the antibodies of the invention are useful to treatrheumatoid arthritis. RA is characterized by inflammation of multiplejoints, cartilage loss and bone erosion that leads to joint destructionand ultimately reduced joint function. Additionally, since RA is asystemic disease, it can have effects in other tissues such as thelungs, eyes and bone marrow. Fewer than 50 percent of patients who havehad RA for more than 10 years can continue to work or function normallyon a day-to-day basis.

The antibodies can be used as first-line therapy in patients with earlyRA (i.e., methotrexate (MTX) naive) and as monotherapy, or incombination with, e.g., MTX or cyclophosphamide. Or, the antibodies canbe used in treatment as second-line therapy for patients who were DMARDand/or MTX refractory, and as monotherapy or in combination with, e.g.,MTX. The humanized BR3 binding antibodies are useful to prevent andcontrol joint damage, delay structural damage, decrease pain associatedwith inflammation in RA, and generally reduce the signs and symptoms inmoderate to severe RA. The RA patient can be treated with the humanizedBR3 antibody prior to, after or together with treatment with other drugsused in treating RA (see combination therapy below). In one embodiment,patients who had previously failed disease-modifying antirheumatic drugsand/or had an inadequate response to methotrexate alone are treated witha humanized BR3 binding antibody of the invention. In one embodiment ofthis treatment, the patients are in a 17-day treatment regimen receivinghumanized BR3 binding antibody alone (1g iv infusions on days 1 and 15);BR3 binding antibody plus cyclophosphamide (750 mg iv infusion days 3and 17); or BR3 binding antibody plus methotrexate.

One method of evaluating treatment efficacy in RA is based on AmericanCollege of Rheumatology (ACR) criteria, which measures the percentage ofimprovement in tender and swollen joints, among other things. The RApatient can be scored at for example, ACR 20 (20 percent improvement)compared with no antibody treatment (e.g baseline before treatment) ortreatment with placebo. Other ways of evaluating the efficacy ofantibody treatment include X-ray scoring such as the Sharp X-ray scoreused to score structural damage such as bone erosion and joint spacenarrowing. Patients can also be evaluated for the prevention of orimprovement in disability based on Health Assessment Questionnaire [HAQ]score, AIMS score, SF-36 at time periods during or after treatment. TheACR 20 criteria may include 20% improvement in both tender (painful)joint count and swollen joint count plus a 20% improvement in at least 3of 5 additional measures:

-   -   1. patient's pain assessment by visual analog scale (VAS),    -   2. patient's global assessment of disease activity (VAS),    -   3. physician's global assessment of disease activity (VAS),    -   4. patient's self-assessed disability measured by the Health        Assessment Questionnaire, and    -   5. acute phase reactants, CRP or ESR.        The ACR 50 and 70 are defined analogously. Preferably, the        patient is administered an amount of a BR3 binding antibody of        the invention effective to achieve at least a score of ACR 20,        preferably at least ACR 30, more preferably at least ACR50, even        more preferably at least ACR70, most preferably at least ACR 75        and higher.

Psoriatic arthritis has unique and distinct radiographic features. Forpsoriatic arthritis, joint erosion and joint space narrowing can beevaluated by the Sharp score as well. The humanized BR3 bindingantibodies of the invention can be used to prevent the joint damage aswell as reduce disease signs and symptoms of the disorder.

Yet another aspect of the invention is a method of treating Lupus or SLEby administering to the patient suffering from SLE, a therapeuticallyeffective amount of a BR3 binding antibody of the invention. SLEDAIscores provide a numerical quantitation of disease activity. The SLEDAIis a weighted index of 24 clinical and laboratory parameters known tocorrelate with disease activity, with a numerical range of 0-103. seeBryan Gescuk & John Davis, “Novel therapeutic agent for systemic lupuserythematosus” in Current Opinion in Rheumatology 2002, 14:515-521.Antibodies to double-stranded DNA are believed to cause renal flares andother manifestations of lupus. Patients undergoing antibody treatmentcan be monitored for time to renal flare, which is defined as asignificant, reproducible increase in serum creatinine, urine protein orblood in the urine. Alternatively or in addition, patients can bemonitored for levels of antinuclear antibodies and antibodies todouble-stranded DNA. Treatments for SLE include high-dosecorticosteroids and/or cyclophosphamide (HDCC).

Spondyloarthropathies are a group of disorders of the joints, includingankylosing spondylitis, psoriatic arthritis and Crohn's disease.Treatment success can be determined by validated patient and physicianglobal assessment measuring tools.

Various medications are used to treat psoriasis; treatment differsdirectly in relation to disease severity. Patients with a more mild formof psoriasis typically utilize topical treatments, such as topicalsteroids, anthralin, calcipotriene, clobetasol, and tazarotene, tomanage the disease while patients with moderate and severe psoriasis aremore likely to employ systemic (methotrexate, retinoids, cyclosporine,PUVA and UVB) therapies. Tars are also used. These therapies have acombination of safety concerns, time consuming regimens, or inconvenientprocesses of treatment. Furthermore, some require expensive equipmentand dedicated space in the office setting. Systemic medications canproduce serious side effects, including hypertension, hyperlipidemia,bone marrow suppression, liver disease, kidney disease andgastrointestinal upset. Also, the use of phototherapy can increase theincidence of skin cancers. In addition to the inconvenience anddiscomfort associated with the use of topical therapies, phototherapyand systemic treatments require cycling patients on and off therapy andmonitoring lifetime exposure due to their side effects.

Treatment efficacy for psoriasis is assessed by monitoring changes inclinical signs and symptoms of the disease including Physician's GlobalAssessment (PGA) changes and Psoriasis Area and Severity Index (PASI)scores, Psoriasis Symptom Assessment (PSA), compared with the baselinecondition. The patient can be measured periodically throughout treatmenton the Visual analog scale used to indicate the degree of itchingexperienced at specific time points.

Patients may experience an infusion reaction or infusion-relatedsymptoms with their first infusion of a therapeutic antibody. Thesesymptoms vary in severity and generally are reversible with medicalintervention. These symptoms include but are not limited to, flu-likefever, chills/rigors, nausea, urticaria, headache, bronchospasm,angioedema. It would be desirable for the disease treatment methods ofthe present invention to minimize infusion reactions. Thus, anotheraspect of the invention is a method of treating the diseases disclosedby administering a BR3 binding antibody wherein the antibody has reducedor no complement dependent cytotoxicity.

Dosage

Depending on the indication to be treated and factors relevant to thedosing that a physician of skill in the field would be familiar with,the antibodies of the invention will be administered at a dosage that isefficacious for the treatment of that indication while minimizingtoxicity and side effects. For the treatment of a cancer, an autoimmunedisease or an immunodeficiency disease, the therapeutically effectivedosage can be in the range of 50 mg/dose to 2.5 g/m². In one embodiment,the dosage administered is about 250 mg/m² to about 400 mg/m² or 500mg/m². In another embodiment, the dosage is about 250-375 mg/m². In yetanother embodiment, the dosage range is 275-375 mg/m².

In one embodiment of the treatment of a BR3 positive B cell neoplasmdescribed herein (e.g., chronic lymphocytic leukemia (CLL), non-Hodgkinslymphoma (NHL), follicular lymphoma (FL) or multiple myeloma), theantibody is administered at a range of 50 mg/dose to 2.5 g/m². For thetreatment of patients suffering from B-cell lymphoma such asnon-Hodgkins lymphoma, in a specific embodiment, the anti-BR3 antibodiesand humanized anti-BR3 antibodies of the invention will be administeredto a human patient at a dosage of 10 mg/kg or 375 mg/m². For treatingNHL, one dosing regimen would be to administer one dose of the antibodycomposition a dosage of 10 mg/kg in the first week of treatment,followed by a 2 week interval, then a second dose of the same amount ofantibody is administered. Generally, NHL patients can receive suchtreatment once during a year but upon recurrence of the lymphoma, suchtreatment can be repeated. In another dosing regimen, patients treatedwith low-grade NHL receive four weeks of an anti-BR3 antibody (375 mg/m2weekly) followed at week five by three additional courses of theantibody plus standard CHOP (cyclophosphamide, doxorubicin, vincristineand prednisone) or CVP (cyclophosphamide, vincristine, prednisone)chemotherapy, which was given every three weeks for three cycles.

For treating rheumatoid arthritis, in one embodiment, the dosage rangefor the anti-BR3 antibody is 125 mg/m² (equivalent to about 200 mg/dose)to 600 mg/m², given in two doses, e.g., the first dose of 200 mg isadministered on day one followed by a second dose of 200 mg on day 15.In different embodiments, the dosage is selected from the groupconsisting of 250 mg/dose, 275 mg/dose, 300 mg/dose, 325 mg/dose, 350mg/dose, 375 mg/dose, 400 mg/dose, 425 mg/dose, 450 mg/dose, 475mg/dose, 500 mg/dose, 525 mg/dose, 550 mg/dose, 575 mg/dose and 600mg/dose.

In treating disease, the BR3 binding antibodies of the invention can beadministered to the patient chronically or intermittently, as determinedby the physician of skill in the disease.

A patient administered a drug by intravenous infusion or subcutaneouslymay experience adverse events such as fever, chills, burning sensation,asthenia and headache. To alleviate or minimize such adverse events, thepatient may receive an initial conditioning dose(s) of the antibodyfollowed by a therapeutic dose. The conditioning dose(s) will be lowerthan the therapeutic dose to condition the patient to tolerate higherdosages.

It is contemplated that BR3 binding antibodies of this invention that(1) lack ADCC function or have reduced ADCC function compared to anantibody comprising a wild type human IgG Fc; (2) lack the ability topartially or fully inhibit BAFF binding to BR3 or (3) lack ADCC functionor have reduced ADCC function compared to an antibody comprising a wildtype human IgG Fc and lack the ability to partially or fully inhibitBAFF binding to BR3 will be useful, for example, as in a replacementtherapy, alternative therapy or a maintenance therapy for patients thathave or are expected to have significantly adverse responses totherapies with anti-BR3 antibodies that inhibit BAFF and BR3 binding andhave ADCC function. For example, it is contemplated that a patient canbe first treated with anti-BR3 antibodies that inhibit BAFF and BR3binding and have ADCC function followed by treatments with anti-BR3antibodies that (1) lack ADCC function or have reduced ADCC functioncompared to antibodies-comprising wild type human IgG Fc; (2) lack theability to partially or fully inhibit BAFF binding to BR3 or (3) lackADCC function or have reduced ADCC function compared to antibodiescomprising wild type human IgG Fc and lack the ability to partially orfully inhibit BAFF binding to BR3.

Route of Administration

The BR3 binding antibodies are administered to a human patient in accordwith known methods, such as by intravenous administration, e.g., as abolus or by continuous infusion over a period of time, by subcutaneous,intramuscular, intraperitoneal, intracerobrospinal, intra-articular,intrasynovial, intrathecal, or inhalation routes, generally byintravenous or subcutaneous administration.

In on embodiment, the anti-BR3 antibody is administered by intravenousinfusion with 0.9% sodium chloride solution as an infusion vehicle. Inanother embodiment, the anti-BR3 antibodies are administered with apre-filled syringe.

Combination Therapy

The BR3-binding antibodies or polypeptides of this invention can be usedin combination with a second therapeutic agent to treat the dease. Itshould be understood that the term second therapeutic agent does notpreclude treating the subjects other additional therapies. The referenceto a second therapeutic agent is meant to differentiate the agent fromthe specific BR3-binding antibody or polypeptide also being used. In oneembodiment, a patient to be treated with the BR3 binding antibodies orpolypeptides for an autoimmune disease or a cancer can be treatedconcurrently, sequentially (before or after), or alternatingly with abiologic response modifier (BRM) to stimulate or restore the ability ofthe immune system to fight disease and/or infection in a multidrugregimen. BRMs can include monoclonal antibodies, such as antibodies thattarget TNF-alpha or IL-1 (e.g., Enbrel®, Remicade®, and Humira®),interferon, interleukins (e.g, IL-2, IL-12) and various types ofcolony-stimulating factors (CSF, GM-CSF, G-CSF). For example, the BRMsmay interfere with inflammatory activity, ultimately decreasing jointdamage.

In one embodiment, the second therapeutic is an IAP inhibitor.

In another embodiment, a patient to be treated with the BR3 bindingantibodies or polypeptides for an autoimmune disease or a cancer can betreated concurrently, sequentially (before or after), or alternatinglywith a B cell depleting agent.

In one embodiment, a patient to be treated with the BR3 bindingantibodies for an autoimmune disease or a cancer can be treatedconcurrently, sequentially (before or after), or alternatingly with aBAFF antagonist.

In another embodiment, the cancers and neoplasms described above, thepatient can be treated with the BR3 binding antibodies of the presentinvention in conjunction with one or more therapeutic agents such as achemotherapeutic agent in a multidrug regimen. The BR3 binding antibodycan be administered concurrently, sequentially (before or after), oralternating with the chemotherapeutic agent, or after non-responsivenesswith other therapy. Standard chemotherapy for lymphoma treatment mayinclude cyclophosphamide, cytarabine, melphalan and mitoxantrone plusmelphalan. CHOP is one of the most common chemotherapy regimens fortreating Non-Hodgkin's lymphoma. The following are the drugs used in theCHOP regimen: cyclophosphamide (brand names cytoxan, neosar); adriamycin(doxorubicin/hydroxydoxorubicin); vincristine (Oncovin); andprednisolone (sometimes called Deltasone or Orasone). In particularembodiments, the BR3 binding antibody is administered to a patient inneed thereof in combination with one or more of the followingchemotherapeutic agents of doxorubicin, cyclophosphamide, vincristineand prednisolone. In a specific embodiment, a patient suffering from alymphoma (such as a non-Hodgkin's lymphoma) is treated with an anti-BR3antibody of the present invention in conjunction with CHOP(cyclophosphamide, doxorubicin, vincristine and prednisone) therapy. Inanother embodiment, a cancer or neoplasm in a patient can be treatedwith a BR3 binding antibody of the invention in combination with CVP(cyclophosphamide, vincristine, and prednisone) chemotherapy. In aspecific embodiment, the patient suffering from BR3-positive NHL istreated with humanized anti-BR3 antibody in conjunction with CVP. In aspecific embodiment of the treatment of chronic lymphocytic leukemia(CLL,) the BR3 binding antibody is administered in conjunction withchemotherapy with one or more nucleoside analogs, such as fludarabine,Cladribine (2-chlorodeoxyadenosine, 2-CdA[Leustatin]), pentostatin(Nipent), with cyclophosphamide.

In treating the autoimmune diseases or autoimmune related conditionsdescribed above, the patient can be treated with the BR3 bindingantibodies of the present invention in conjunction with a secondtherapeutic agent, such as an immunosuppressive agent, such as in amulti drug regimen. The BR3 binding antibody can be administeredconcurrently, sequentially or alternating with the immunosuppressiveagent or upon non-responsiveness with other therapy. Theimmunosuppressive agent can be administered at the same or lesserdosages than as set forth in the art. The preferred adjunctimmunosuppressive agent will depend on many factors, including the typeof disorder being treated as well as the patient's history.

“Immunosuppressive agent” as used herein for adjunct therapy refers tosubstances that act to suppress or mask the immune system of a patient.Such agents would include substances that suppress cytokine production,down regulate or suppress self-antigen expression, or mask the MHCantigens. Examples of such agents include steroids such asglucocorticosteroids, e.g., prednisone, methylprednisolone, anddexamethasone; 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat.No. 4,665,077), azathioprine (or cyclophosphamide, if there is anadverse reaction to azathioprine); bromocryptine; glutaraldehyde (whichmasks the MHC antigens, as described in U.S. Pat. No. 4,120,649);anti-idiotypic antibodies for MHC antigens and MHC fragments;cyclosporin A; cytokine or cytokine receptor antagonists includinganti-interferon

,

, or

antibodies; anti-tumor necrosis factor-antibodies; anti-tumor necrosisfactor-antibodies; anti-interleukin-2 antibodies and anti-IL-2 receptorantibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin;pan-T antibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies;soluble peptide containing a LFA-3 binding domain (WO 90/08187 publishedJul. 26, 1990); streptokinase; TGF

; streptodornase; RNA or DNA from the host; FK506; RS-61443;deoxyspergualin; rapamycin; T-cell receptor (U.S. Pat. No. 5,114,721);T-cell receptor fragments (Offner et al., Science 251:430-432 (1991); WO90/11294; and WO 91/01133); and T cell receptor antibodies (EP 340,109)such as T10B9.

For the treatment of rheumatoid arthritis, the patient can be treatedwith a BR3 antibody of the invention in conjunction with any one or moreof the following drugs: DMARDS (disease-modifying anti-rheumatic drugs(e.g., methotrexate), NSAI or NSAID (non-steroidal anti-inflammatorydrugs), HUMIRA® (adalimumab; Abbott Laboratories), ARAVA® (leflunomide),REMICADE® (infliximab; Centocor Inc., of Malvern, Pa.), ENBREL®(etanercept; Immunex, Wash.), COX-2 inhibitors. DMARDs commonly used inRA are hydroxycloroquine, sulfasalazine, methotrexate, leflunomide,etanercept, infliximab, azathioprine, D-penicillamine, Gold (oral), Gold(intramuscular), minocycline, cyclosporine, Staphylococcal protein Aimmunoadsorption. Adalimumab is a human monoclonal antibody that bindsto TNF. Infliximab is a chimeric monoclonal antibody that binds to TNF.Etanercept is an “immunoadhesin” fusion protein consisting of theextracellular ligand binding portion of the human 75 kD (p75) tumornecrosis factor receptor (TNFR) linked to the Fc portion of a humanIgG1. For conventional treatment of RA, see, e.g., “Guidelines for themanagement of rheumatoid arthritis” Arthritis & Rheumatism 46(2):328-346 (February, 2002). In a specific embodiment, the RA patient istreated with a BR3 antibody of the invention in conjunction withmethotrexate (MTX). An exemplary dosage of MTX is about 7.5-25 mg/kg/wk.MTX can be administered orally and subcutaneously.

For the treatment of ankylosing spondylitis, psoriatic arthritis andCrohn's disease, the patient can be treated with a BR3 binding antibodyof the invention in conjunction with, for example, Remicade®(infliximab; from Centocor Inc., of Malvern, Pa.), ENBREL® (etanercept;Immunex, Wash.).

Treatments for SLE include high-dose corticosteroids and/orcyclophosphamide (HDCC).

For the treatment of psoriasis, patients can be administered a BR3binding antibody in conjunction with topical treatments, such as topicalsteroids, anthralin, calcipotriene, clobetasol, and tazarotene, or withmethotrexate, retinoids, cyclosporine, PUVA and UVB therapies. In oneembodiment, the psoriasis patient is treated with the BR3 bindingantibody sequentially or concurrently with cyclosporine.

Pharmaceutical Formulations

Therapeutic formulations of the BR3-binding antibodies used inaccordance with the present invention are prepared for storage by mixingan antibody having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(sRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.(1980)), in the form of lyophilized formulations or aqueous solutions.Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such asolyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine,histidine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugars such as sucrose, mannitol, trehalose orsorbitol; salt-forming counter-ions such as sodium; metal complexes(e.g. Zn-protein complexes); and/or non-ionic surfactants such asTWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Exemplary anti-BR3 antibody formulations are described in WO98/56418,expressly incorporated herein by reference. Another formulation is aliquid multidose formulation comprising the anti-BR3 antibody at 40mg/mL, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02%polysorbate 20 at pH 5.0 that has a minimum shelf life of two yearsstorage at 2-8° C. Another anti-BR3 formulation of interest comprises 10mg/mL antibody in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citratedihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection, pH6.5. Yet another aqueous pharmaceutical formulation comprises 10-30 mMsodium acetate from about pH 4.8 to about pH 5.5, preferably at pH5.5,polysorbate as a surfactant in a an amount of about 0.01-0.1% v/v,trehalose at an amount of about 2-10% w/v, and benzyl alcohol as apreservative (U.S. Pat. No. 6,171,586). Lyophilized formulations adaptedfor subcutaneous administration are described in WO97/04801. Suchlyophilized formulations may be reconstituted with a suitable diluent toa high protein concentration and the reconstituted formulation may beadministered subcutaneously to the mammal to be treated herein.

One formulation for the humanized anti-BR3 antibody is antibody at 12-14mg/mL in 10 mM histidine, 6% sucrose, 0.02% polysorbate 20, pH 5.8.

In a specific embodiment, anti-BR3 antibody and in particular 9.1RF,9.1RF (N434 mutants), or V3-46s is formulated at 20 mg/mL antibody in 10mM histidine sulfate, 60 mg/mL sucrose., 0.2 mg/ml polysorbate 20, andSterile Water for Injection, at pH5.8.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide a cytotoxic agent,chemotherapeutic agent, cytokine or immunosuppressive agent (e.g. onewhich acts on T cells, such as cyclosporin or an antibody that binds Tcells, e.g. one which binds LFA-1). The effective amount of such otheragents depends on the amount of antibody present in the formulation, thetype of disease or disorder or treatment, and other factors discussedabove. These are generally used in the same dosages and withadministration routes as described herein or about from 1 to 99% of theheretofore employed dosages.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antagonist, which matrices are inthe form of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Articles of Manufacture and Kits

Another embodiment of the invention is an article of manufacturecontaining materials useful for the treatment of autoimmune diseases andrelated conditions and BR3 positive cancers such as non-Hodgkin'slymphoma. Yet another embodiment of the invention is an article ofmanufacture containing materials useful for the treatment ofimmunodeficiency diseases. The article of manufacture comprises acontainer and a label or package insert on or associated with thecontainer. Suitable containers include, for example, bottles, vials,syringes, etc. The containers may be formed from a variety of materialssuch as glass or plastic. The container holds a composition which iseffective for treating the condition and may have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is a BR3 binding antibody of theinvention. The label or package insert indicates that the composition isused for treating the particular condition. The label or package insertwill further comprise instructions for administering the antibodycomposition to the patient. Articles of manufacture and kits comprisingcombinatorial therapies described herein are also contemplated.

Package insert refers to instructions customarily included in commercialpackages of therapeutic products, that contain information about theindications, usage, dosage, administration, contraindications and/orwarnings concerning the use of such therapeutic products. In oneembodiment, the package insert indicates that the composition is usedfor treating non-Hodgkins' lymphoma.

Additionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

Kits are also provided that are useful for various purposes, e.g., forB-cell killing assays, as a positive control for apoptosis assays, forpurification or immunoprecipitation of BR3 from cells. For isolation andpurification of BR3, the kit can contain an anti-BR3 antibody coupled tobeads (e.g., sepharose beads). Kits can be provided which contain theantibodies for detection and quantitation of BR3 in vitro, e.g. in anELISA or a Western blot. As with the article of manufacture, the kitcomprises a container and a label or package insert on or associatedwith the container. The container holds a composition comprising atleast one anti-BR3 antibody of the invention. Additional containers maybe included that contain, e.g., diluents and buffers, controlantibodies. The label or package insert may provide a description of thecomposition as well as instructions for the intended in vitro ordiagnostic use.

Monoclonal Antibodies

Anti-BR3 antibodies can be monoclonal antibodies. Monoclonal antibodiescan be prepared, e.g., using hybridoma methods, such as those describedby Kohler and Milstein, Nature, 256:495 (1975) or can be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567) or can be produced bythe methods described herein in the Example section. In a hybridomamethod, a mouse, hamster, or other appropriate host animal is typicallyimmunized with an immunizing agent to elicit lymphocytes that produce orare capable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes can be immunized invitro.

The immunizing agent will typically include the BR3 polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell. Goding, Monoclonal Antibodies: Principles and Practice (New York:Academic Press, 1986), pp. 59-103. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine, and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells can be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high-level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies. Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications (MarcelDekker, Inc.: New York, 1987) pp. 51-63.

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theBR3 polypeptide. Preferably, the binding specificity of monoclonalantibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.Goding, supra. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells can be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies can also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also can be modified, for example, bysubstituting the coding sequence for human heavy- and light-chainconstant domains in place of the homologous murine sequences (U.S. Pat.No. 4,816,567; Morrison et al., supra) or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies can be monovalent antibodies. Methods for preparingmonovalent antibodies are known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy-chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using techniques known in the art.

Human and Humanized Antibodies

The anti-BR3 antibodies can further comprise humanized antibodies orhuman antibodies. Humanized forms of non-human (e.g., murine) antibodiesare chimeric immunoglobulins, immunoglobulin chains, or fragmentsthereof (such as Fv, Fab, Fab′, F(ab′)₂, or other antigen-bindingsubsequences of antibodies) that typically contain minimal sequencederived from non-human immunoglobulin. Humanized antibodies includehuman immunoglobulins (recipient antibody) in which residues from a CDRof the recipient are replaced by residues from a CDR of a non-humanspecies (donor antibody) such as mouse, rat, or rabbit having thedesired specificity, affinity, and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies can also compriseresidues that are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin, and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody preferably also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin. Jones et al., Nature, 321: 522-525(1986); Riechmann et al., Nature, 332: 323-329 (1988); Presta, Curr. Op.Struct. Biol., 2:593-596 (1992).

Some methods for humanizing non-human antibodies are described in theart and below in the Examples. Generally, a humanized antibody has oneor more amino acid residues introduced into it from a source that isnon-human. These non-human amino acid residues are often referred to as“import” residues, which are typically taken from an “import” variabledomain. According to one embodiment, humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327(1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare antibodies (U.S. Pat. No. 4,816,567), wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some-CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array into such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. See, e.g.,Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Yearin Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669(all of GenPharm); 5,545,807; and WO 97/17852. Alternatively, humanantibodies can be made by introducing human immunoglobulin loci intotransgenic animals, e.g., mice in which the endogenous immunoglobulingenes have been partially or completely inactivated. Upon challenge,human antibody production is observed that closely resembles that seenin humans in all respects, including gene rearrangement, assembly, andantibody repertoire. This approach is described, for example, in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and5,661,016, and in the following scientific publications: Marks et al.,Bio/Technology, 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859(1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al., NatureBiotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995).Alternatively, phage display technology (McCafferty et al., Nature348:552-553 [1990]) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to one embodiment of thistechnique, antibody V domain sequences are cloned in-frame into either amajor or minor coat protein gene of a filamentous bacteriophage, such asM13 or fd, and displayed as functional antibody fragments on the surfaceof the phage particle. Phage display can be performed in a variety offormats, e.g., as described below in the Examples section or as reviewedin, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion inStructural Biology 3:564-571 (1993). Several sources of V-gene segmentscan be used for phage display. Clackson et al., Nature, 352:624-628(1991) isolated a diverse array of anti-oxazolone antibodies from asmall random combinatorial library of V genes derived from the spleensof immunized mice. A repertoire of V genes from unimmunized human donorscan be constructed and antibodies to a diverse array of antigens(including self-antigens) can be isolated essentially following thetechniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991),or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos.5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries. Hoogenboom and Winter, J.Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581(1991). The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies. Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1): 86-95 (1991).

Multi-Specific Anti-BR3 Antibodies

Multi-specific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for two or more differentantigens (e.g., bispecific antibodies have binding specificities for atleast two antigens). For example, one of the binding specificities canbe for the BR3 polypeptide, the other one can be for any other antigen.According to one preferred embodiment, the other antigen is acell-surface protein or receptor or receptor subunit. For example, thecell-surface protein can be a natural killer (NK) cell receptor. Thus,according to one embodiment, a bispecific antibody of this invention canbind BR3 and bind a NK cell and, optionally, activate the NK cell.

Examples of methods for making bispecific antibodies have beendescribed. Traditionally, the recombinant production of bispecificantibodies is based on the co-expression of two immunoglobulinheavy-chain/light-chain pairs, where the two heavy chains have differentspecificities. Milstein and Cuello, Nature 305: 537-539 (1983). Becauseof the random assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of ten differentantibody molecules, of which only one has the correct bispecificstructure. The purification of the correct molecule is usuallyaccomplished by affinity chromatography steps. Similar procedures aredisclosed in WO 93/08829, published 13 May 1993, and in Traunecker etal., EMBO J., 10: 3655-3659 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant-domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies, see, for example,Suresh et al., Methods in Enzymology, 121: 210 (1986).

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Iimnunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise a VHconnected to a VL by a linker which is too short to allow pairingbetween the two domains on the same chain. Accordingly, the VH and VLdomains of one fragment are forced to pair with the complementary VL andVH domains of another fragment, thereby forming two antigen-bindingsites. Another strategy for making bispecific antibody fragments by theuse of single-chain Fv (sFv) dimers has also been reported. See Gruberet al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

Heteroconjugate Antibodies

Heteroconjugate antibodies are composed of two covalently joinedantibodies. Such antibodies have, for example, been proposed to targetimmune-system cells to unwanted cells (U.S. Pat. No. 4,676,980), and fortreatment of HIV infection. WO 91/00360; WO 972/200373; EP 03089. It iscontemplated that the antibodies can be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins can be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.Pat. No. 4,676,980.

Engineering of Afucosylated Antibodies and Underfucosylated AntibodyCompositions

The invention herein relates to a method for making an afucosylatedanti-BR3 antibody or an underfucosylated composition of anti-BR3antibodies that includes a substantially homogeneous preparation of Fcregion-containing antibodies, wherein about 20-100% of the antibodies inthe composition comprises a mature core carbohydrate lacking fucose,attached to the Fc region of the antibody. The antibodies can beprepared, for example, by (a) use of an engineered or mutant host cellthat is deficient in fucose metabolism such that it has a reducedability (or is unable to) fucosylate proteins expressed therein; (b)culturing cells under conditions which prevent or reduce fucosylation;(c) post-translational removal of fucose (e.g. with a fucosidaseenzyme); (d) post-translational addition of the desired carbohydrate,e.g. after recombinant expression of a non-glycosylated antibody; or (e)purification of the antibody so as to select for product which is notfucosylated. The present invention contemplates combining two or more ofthese exemplary methods (a)-(e).

According to one preferred embodiment, nucleic acid encoding the desiredantibody is expressed in a host cell that has a reduced ability (or isunable to) fucosylate proteins expressed therein. For example, the hostcell can be a dihydrofolate reductase (DHFR) deficient chinese hamsterovary (CHO) cell, e.g. a Lec13 CHO cell, or e.g., a CHO-K1, DUX-B11,CHO-DP12 or CHO-DG44 CHO host cell which has been modified so that theantibody produced therein is not substantially fucosylated. Thus, thecell may display altered expression or activity for thefucosyltransferase enzyme, or another enzyme or substrate involved inadding fucose to the N-linked oligosaccharide may have diminishedactivity and/or reduced levels in the host cell. Alternately, the hostcell can be transfected with a vector producing an RNAi targeting aprotein in the fucosyl pathway.

The core carbohydrate structure is mature, thus, the use of inhibitors,such as castanospermine, which inhibit or interfere with processing ofthe mature carbohydrate should generally be avoided. According to oneembodiment, anywhere from 1-100% of the anti-BR3 antibodies areafucosylated. According to one preferred embodiment of the invention, anunderfucosylated composition of antibodies is recovered wherein about20-100% of the antibody in the composition recovered from therecombinant host cell producing the antibody will have a corecarbohydrate structure which lacks fucose attached to the Fc region ofthe antibody, hereinafter a “fucose-free antibody composition.” By“recovered” here is meant that material obtained directly from the hostcell culture without subjecting that material to a purification stepwhich enriches for fucose-free antibody.

However, the present invention does contemplate enriching the amount offucose-free antibody by various techniques, such as purification using alectin substrate to remove fucose-containing antibody from the desiredcomposition.

It will be appreciated that the amount of fucose-free antibody fromvarious batches of recombinantly produced antibody may vary. In onepreferred embodiment, about 90-99% of the antibody in the compositioncomprises a mature core carbohydrate structure which lacks fucoseattached to the Fc region of the antibody.

Various forms of the carbohydrate structure may exist in the composition(see FIG. 7). For instance, the carbohydrate attached to the antibodymay be represented by the following formula:

wherein,M is mannose.

GN is GlcNAc.

X₁ is an optional bisecting GlcNAc residue, with additionalmonosaccharide(s) optionally attached to the bisecting GlcNAc.X₂ is a preferred GlcNAc residue.X₃ is an optional Gal residue, one Gal residue may be attached to eachGN arm.X₄ is an optional terminal sialic acid residues, one or two sialic acidresidues may be attached.

The fucose-free antibody compositions herein display improved binding toone or more FcγRIII receptors, compared to a composition of the sameantibody, but where most (e.g. about 50-100%, or about 70-100%) of theantibody in that composition has fucose attached to the mature corecarbohydrate structure (hereinafter a “fucose-containing antibodycomposition). For instance, the fucose-free antibody compositions hereinmay display 3-1000 fold improved binding to an FcγRIII, such as FcγRIII(F158), when compared to the fucose-containing antibody composition. Inthat the F158 allotype is less effective in interacting with human IgGthan V158, this is thought to provide a significant advantage from atherapeutic perspective, especially in patients who express FcγRIII(F158). Moreover, the fucose-free antibody compositions herein displaybetter ADCC activity compared to their counterpart fucose-containingantibody compositions, e.g. from about 2-20 fold improved ADCC activity.

Aside from the fucose-free mature core carbohydrate structure,additional oligosaccharides may be attached to the core carbohydratestructure. For instance, a bisecting GlcNAc may, or may not be,attached. By the way of example, the host cell may lack the GnTIIIenzyme and hence the antibody may be essentially free of bisectingGlcNAc. Alternatively, the antibody may be expressed in a host cell(e.g. a Y0 host or engineered CHO cell) which adds the bisecting GlcNAc.One or more (generally one or two) galactose residues may also beattached to the core carbohydrate structure. Finally, one or moreterminal sialic acid residues (usually one or two) may be attached tocore carbohydrate structure, e.g. by linkage to galactose residue(s).

The compositions herein are, in the preferred embodiment, prepared andintended for therapeutic use. Hence, the preferred composition is apharmaceutical preparation comprising the antibody and apharmaceutically acceptable carrier or diluent such as those exemplifiedbelow. Such preparations are usually sterile and may be lyophilized.

Effector Function Engineering

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) can beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedcan have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See, Caron et al., J. Exp. Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity can also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and can thereby have enhancedcomplement lysis and ADCC capabilities. See, Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

Mutations or alterations in the Fc region sequences can be made toimprove FcR binding (e.g., FcgammaR, FcRn). According to one embodiment,an antibody of this invention has at least one altered effector functionselected from the group consisting of ADCC, CDC, and improved FcRnbinding compared to a native IgG or a parent antibody. Examples ofseveral useful specific mutations are described in, e.g., Shields, R Let al. (2001) JBC 276(6)6591-6604; Presta, L. G., (2002) BiochemicalSociety Transactions 30(4):487-490; and WO publication WO00/42072.

According to one embodiment, the Fc receptor mutation is a substitutionin at least one position selected from the group consisting of: 238,239, 246, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270,272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296,298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329,330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382,388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 of the Fcregion, wherein the numbering of the residues in the Fc region isaccording to the EU numbering system. According to one specificembodiment, the substitution is a 434 residue substitution selected fromthe group consisting of N434A, N434F, N4343Y and N434H. According toanother embodiment, the substitutions are a D265A/N297A mutation.According to another embodiment, the substitutions are S298A/E333A/K334Aor S298A/K326A/E333A/K334A. According to another embodiment, thesubstitution is K322A.

Examples of native sequence human IgG Fc region sequences, humIgG1(non-A and A allotypes) (SEQ ID NOs:133 and 135, respectively), humIgG2(SEQ ID NO:136), humIgG3 (SEQ ID NO:137) and humIgG4 (SEQ ID NO:138)have been described previously. Examples of native sequence murine IgGFc region sequences, murIgG1 (SEQ ID NO:139), murIgG2A (SEQ ID NO:140),murIgG2B (SEQ ID NO:141) and murIgG3 (SEQ ID NO:142), have also beendescribed previously. Example of other mutations may be found inWO2006053301A2, WO2006047350A2, US20060134105A1, WO2005092925A2,US20050244403A1, WO2005077981A2, US20050249723A1, WO2003074679A2,US20040110226A1, WO2004029207A2 US20040132101A, WO2004099249A2,US20050054832A1, WO2006019447A1, US20060024298A1, US20060121032A1.

In addition, various classes of Fc region variants are described belowin Table 3.

TABLE 3 Classes of Fc region variants. Class FcR binding propertyPosition of Fc region substitution(s) 1A reduced binding to all FcγR238, 265, 269, 270, 297*, 327, 329 1B reduced binding to both FcγRII and239, 294, 295, 303, 338, 373, 376, 416, 435 FcγRIII 2 improved bindingto both FcγRII and 256, 290, 312, 326, 330, 339, 378, 430 FcγRIII 3improved binding to FcγRII and no effect 255, 258, 267, 276, 280, 283,285, 286, 305, on FcγRIII binding 307, 309, 315, 320, 331, 337, 398 4improved binding to FcγRII and reduced 268, 272, 301, 322, 340 bindingto FcγRIII 5 reduced binding to FcγRII and no effect 292, 324, 335, 414,419, 438, 439 on FcγRIII binding 6 reduced binding to FcγRII andimproved 298, 333 binding to FcγRIII 7 no effect on FcγRII binding andreduced 248, 249, 252, 254, 278, 289, 293, 296, 338, binding to FcγRIII382, 388, 389, 434, 437 8 no effect on FcγRII binding and 334, 360improved binding to FcγRIII *deglycosylated version

Pharmaceutical Compositions of Antibodies and Polypeptides

Antibodies specifically binding a BR3 polypeptide identified herein, aswell as other molecules identified by the screening assays disclosedhereinbefore, can be administered for the treatment of various disordersas noted above and below in the form of pharmaceutical compositions.

Lipofectins or liposomes can be used to deliver the polypeptides andantibodies or compositions of this invention into cells. Liposomescontaining the antibody are prepared by methods known in the art, suchas described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688(1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); andU.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhancedcirculation time are disclosed in U.S. Pat. No. 5,013,556. Particularlyuseful liposomes can be generated by the reverse-phase evaporationmethod with a lipid composition comprising phosphatidylcholine,cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE).Liposomes are extruded through filters of defined pore size to yieldliposomes with the desired diameter. Fab′ fragments of the antibody ofthe present invention can be conjugated to the liposomes as described inMartin et al., J. Biol. Chem., 257: 286-288 (1982) via adisulfide-interchange reaction. A chemotherapeutic agent (such asDoxorubicin) is optionally contained within the liposome. See, Gabizonet al., J. National Cancer Inst., 81(19): 1484 (1989).

Where antibody fragments are used, the smallest inhibitory fragment thatspecifically binds to the binding domain of the target protein ispreferred. For example, based upon the variable-region sequences of anantibody, peptide molecules can be designed that retain the ability tobind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology. See, e.g.,Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).

The formulation herein can also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition can comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,chemotherapeutic agent, or growth-inhibitory agent. Such molecules aresuitably present in combination in amounts that are effective for thepurpose intended.

The active ingredients can also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they can denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization canbe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Diagnostic Use and Imaging

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a BR3 can be used for diagnostic purposes todetect, diagnose, or monitor diseases and/or disorders associated withthe expression, aberrant expression and/or activity of a polypeptide ofthe invention. According to one preferred embodiment, the anti-BR3antibodies used in diagnostic assays or imaging assays that involveinjection of the anti-BR3 antibody into the subject are antibodies thatdo not block the interaction between BAFF and BR3 or only partiallyblocks the interation between BAFF and BR3. The invention provides forthe detection of aberrant expression of a BR3 polypeptide, comprising(a) assaying the expression of the BR3 polypeptide in cells or bodyfluid of an individual using one or more antibodies of this inventionand (b) comparing the level of gene expression with a standard geneexpression level, whereby an increase or decrease in the assayed geneexpression level compared to the standard expression level is indicativeof aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder tobe treated with an anti-BR3 antibody or polypeptide of this invention,comprising (a) assaying the expression of BR3 polypeptide in cells orbody fluid of an individual using an antibody of this invention, (b)assaying the expression of BAFF polypeptide in cells or body fluid ofthe individual and (c) comparing the level of BAFF gene expression witha standard gene expression level, whereby an increase or decrease in theassayed BAFF gene expression level compared to the standard expressionlevel and the presence of BR3 polypeptide in the fluid or diseasedtissue is indicative of a disorder to be treated with an anti-BR3antibody or polypeptide. With respect to cancer, the presence of BR3 ora relatively high amount of BR3 transcript in biopsied tissue from anindividual may indicate a predisposition for the development of thedisease, or may provide a means for detecting the disease prior to theappearance of actual clinical symptoms. A more definitive diagnosis ofthis type may allow health professionals to employ preventative measuresor aggressive treatment earlier thereby preventing the development orfurther progression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon(¹⁴C), sulfur (³⁵S), tritium (³H), indium (^(115m)In, ^(113m)In ¹¹²In,¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium(⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe),fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y_(,)⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru; lmmol; and fluorescent labels,such as fluorescein and rhodamine, and biotin.

Techniques known in the art may be applied to label antibodies of theinvention. Such techniques include, but are not limited to, the use ofbifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065;5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990;5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contentsof each of which are hereby incorporated by reference in its entirety).

Diagnosis of a disease or disorder associated with expression oraberrant expression of a BR3 molecule in an animal, preferably a mammaland most preferably a human can comprise the step of detecting BR3molecules in the mammal. In one embodiment, diagnosis comprises: (a)administering (for example, parenterally, subcutaneously, orintraperitoneally) to a mammal an effective amount of a labeled anti-BR3antibody or polypeptide which specifically binds to the BR3 molecule,respectively; (b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the BR3 molecule is expressed(and for unbound labeled molecule to be cleared to background level);(c) determining background level; and (d) detecting the labeled moleculein the subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with expression or aberrant expression of BR3.Background level can be determined by various methods including,comparing the amount of labeled molecule detected to a standard valuepreviously determined for a particular system. According to specificembodiments, the antibodies of the invention are used to quantitate orqualitate concentrations of cells of B cell lineage or cells ofmonocytic lineage.

According to one specific embodiment, BR3 polypeptide expression oroverexpression is determined in a diagnostic or prognostic assay byevaluating levels of BR3 present on the surface of a cell, or secretedby the cell (e.g., via an immunohistochemistry assay using anti-BR3antibodies or anti-BAFF antibodies; FACS analysis, etc.). Alternatively,or additionally, one can measure levels of BR3 polypeptide-encodingnucleic acid or mRNA in the cell, e.g., via fluorescent in situhybridization using a nucleic acid based probe corresponding to aBR3-encoding nucleic acid or the complement thereof; (FISH; seeWO98/45479 published October, 1998), Southern blotting, Northernblotting, or polymerase chain reaction (PCR) techniques, such as realtime quantitative PCR (RT-PCR). One can also study. BR3 molecules orBAFF molecules overexpression by measuring shed antigen in a biologicalfluid such as serum, e.g., using antibody-based assays (see also, e.g.,U.S. Pat. No. 4,933,294 issued Jun. 12, 1990; WO91/05264 published Apr.18, 1991; U.S. Pat. No. 5,401,638 issued Mar. 28, 1995; and Sias et al.,J. Immunol. Methods 132:73-80 (1990)). Aside from the above assays,various in vivo assays are available to the skilled practitioner. Forexample, one can expose cells within the body of the mammal to anantibody which is optionally labeled with a detectable label, e.g., aradioactive isotope, and binding of the antibody to cells in the mammalcan be evaluated, e.g., by external scanning for radioactivity or byanalyzing a biopsy taken from a mammal previously exposed to theantibody.

Assays

All publications (including patents and patent applications) citedherein are hereby incorporated in their entirety by reference, includingU.S. Provisional Application No. 60/640,323, filed Dec. 31, 2004.

The following DNA sequences were deposited under the terms of theBudapest Treaty with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209, USA as described below:

Material Deposit No. Deposit Date Hu9.1-RF-H-IgG PTA-6315 Nov. 17, 2004Hu9.1-RF-L-IgG PTA-6316 Nov. 17, 2004 Hu2.1-46.DANA-H-IgG PTA-6313 Nov.17, 2004 Hu2.1-46.DANA-L-IgG PTA-6314 Nov. 17, 2004 HuV3-46s-H-IgGPTA-6317 Nov. 17, 2004 HuV3-46s-L-IgG PTA-6318 Nov. 17, 2004 Murine BCells: 12B12.1 PTA-6624 Apr. 8, 2005 Murine B Cells: 3.1 PTA-6622 Apr.8, 2005

The deposits herein were made under the provisions of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure and the Regulations thereunder(Budapest Treaty). This assures maintenance of a viable culture of thedeposits for 30 years from the date of deposit. The deposits will bemade available by ATCC under the terms of the Budapest Treaty, andsubject to an agreement between Genentech, Inc. and ATCC, which assurespermanent and unrestricted availability of the progeny of the culture ofthe deposits to the public upon issuance of the pertinent U.S. patent orupon laying open to the public of any U.S. or foreign patentapplication, whichever comes first, and assures availability of theprogeny to one determined by the U.S. Commissioner of Patents andTrademarks to be entitled thereto according to 35 U.S.C. 122 and theCommissioner's rules pursuant to thereto (including 37 C.F.R. 1.14 withparticular reference to 8860G 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposits should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

Commercially available reagents referred to in the Examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following Examples, andthroughout the specification, by ATCC accession numbers is the American.Type Culture Collection, Manassas, Va. Unless otherwise noted, thepresent invention uses standard procedures of recombinant DNAtechnology, such as those described hereinabove and in the followingtextbooks: Sambrook et al., supra-, Ausubel et al., Current Protocols inMolecular Biology (Green Publishing Associates and Wiley Interscience,N.Y., 1989); Innis et al., PCR Protocols: A Guide to Methods andApplications (Academic Press, Inc.: N.Y., 1990); Harlow et al.,Antibodies: A Laboratory Manual (Cold Spring Harbor Press: Cold SpringHarbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press: Oxford,1984); Freshney, Animal Cell Culture, 1987; Coligan et al., CurrentProtocols in Immunology, 1991.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

The foregoing written description is considered to be sufficient toenable one skilled in the art to practice the invention. The followingExamples are offered for illustrative purposes only, and are notintended to limit the scope of the present invention in any way. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and fall within the scope of the appendedclaims.

EXAMPLES Example 1 Materials

Hybridomas producing murine monoclonal antibodies referred to as 2.1 and9.1, have been previously described (International Patent ApplicationPCT/US01/28006 (WO 02/24909)) and deposited in the American Type CultureCollection (ATCC) as ATCC NO. 3689 and ATCC NO. 3688, respectively(10801 University Blvd., Manassas, Va. 20110-2209, USA). Hybridomas 3.1and 12B12.1 were deposited as ATCC Deposit PTA-6622 and ATCC DepositPTA-6624, respectively. Other antibodies were made using phage displaytechniques. The sequences of those antibodies are provided herein and/orhave been deposited as described herein.

Example 2 BJAB Cell Binding Assay

BJAB cells, a human Burkitt lymphoma cell line, were cultured in RPMImedia supplemented with 10% FBS, penicillin (100 U/ml, Gibco-Invitrogen,Carlsbad, Calif.), streptomycin (100 μg/ml, Gibco), and L-glutamine (10mM). Analysis of receptor expression by flow cytometry demonstrated thatBJAB cells express high levels of BR3 and undetectable levels of BCMAand TACI. For binding assays, cells were washed with cold assay buffer(phosphate buffered saline (PBS), pH 7.4) containing 1% fetal bovineserum (FBS)). The cell density was adjusted to 1.25×10⁶/ml, and 200 μlof cell suspension was aliquoted into the wells of 96 well round-bottompolypropylene plates (NUNC, Neptune, N.J.; 250,000 cells/well). Theplates containing the cells were centrifuged at 1200 rpm for 5 min at 4°C., and the supernatant was carefully aspirated away from the cellpellets. V3-1m (or mV3-1) and V3-1h refers to the variable region of theV3-1 antibody fused to the constant regions of mouse IgG2a or humanIgG1, respectively. The term chimeric 11G9, chimeric 2.1 or chimeric 9.1refers to the fusion of the variable regions of 11G9, 2.1 or 9.1,respectively, to the constant regions of a human IgG1. For theseexperiments, full length antibodies (IgG) were used.

Direct and competitive binding assays were performed as follows. For thedirect binding assay, IgG antibody samples were serially diluted in coldassay buffer to concentrations ranging between 300-0.02 nM. Samples (100μl) were added to the pelleted cells, and the plates were incubated for45 min on ice. An additional 100 μl assay buffer was then added to eachwell, and the plates were centrifuged at 1200 rpm for 5 min at 4° C.After carefully aspirating the supernatant, the cells were washed twoadditional times with 200 μl assay buffer. An anti-mouse IgG Fc-HRP orgoat anti-human IgG Fc-HRP, as appropriate, was diluted 1/10,000 in coldassay buffer was added (100 μl/well, Jackson ImmunoResearch, West Grove,Pa.), and the plates were incubated on ice for 45 min. Following twowashes with 200 μl cold assay buffer, tetramethyl benzidine (TMB,Kirkegaard & Perry Laboratories, Gaithersburg, Md.) was added, and colorwas allowed to develop for 10 min. One hundred microliters 1 M H₃PO₄ wasadded to stop the reaction. The plates were then read on a microplatereader at 450 nm with a 620 nm reference. In the direct binding assay,the indicated concentrations of mAbs were added to BJAB cells and boundmAb was detected.

In the competitive binding assay, the anti-BR3 mAbs compete withbiotinylated BAFF for binding to cell surface BR3. Human BAFF expressedand purified at Genentech was biotinylated using NHS-X-biotin (ResearchOrganics, Cleveland, Ohio) as previously described (Rodriguez, C. F., etal., (1998) J. Immunol. Methods 219:45-55). The anti-BR3 antibodies wereserially diluted and combined with an equal volume of biotin-BAFF togive final concentrations of 333-0.15 nM mAb and 10 ng/ml biotin-BAFF.The diluted samples were added to the pelleted BJAB cells in 96 wellplates as described above. After 45 min incubation on ice, the cellswere washed twice with 200 μl cold assay buffer, and streptavidin-HRP(AMDEX, Amersham Biosciences, Piscataway, N.J.) diluted 1/5,000 in assaybuffer was added (100 μl/well). The plates were incubated for a final 45min on ice. After washing twice with cold assay buffer, color wasdeveloped using TMB, the reaction was stopped with H₃PO₄, and the plateswere read as described above.

Based on the results of the BJAB binding assays, the antibodies could beclassified as either blocking or non-blocking. In the competitive assay,four mAbs (11G9, 2.1, 9.1, and V3-1) fully blocked binding ofbiotin-BAFF while three others (1E9, 7B2, and 8G4) resulted in partialinhibition. MAbs 1A11, 8E4, 10E2, 12B12 and 3.1 were found to benon-blocking. Of these nonblocking antibodies, 1A11 and 8E4 boundrelatively poorly to the BJABs in the direct binding assay, whilebinding of 10E12 and 12B12 gave somewhat higher maximum signal than theother mAbs. Mouse IgG1, IgG2a, and IgG2b isotype controls showed nodetectable binding to BJABs, and the HRP-conjugated anti-mouse IgG Fcdetection antibody was shown to bind equally to these isotypes. MAbsV3-1m and B9C11 were evaluated in both the BJAB and BHK binding assays.While both of these blocking antibodies bind to murine BR3, only V3-1mbinds to human BR3. Results with V3-1h were similar to those observedfor V3-1m.

Example 3 Epitope Mapping ELISAs

Epitope mapping studies were performed by ELISAs in which dilutioncurves of unlabeled mAbs competed with biotinylated 2.1, 9.1, 11G9, or1E9 for binding to vhBR3-Fc. The results for the fully blocking mAbs(11G9, 2.1, and 9.1) suggested that the epitope for 11G9 binding wasspatially located between the epitopes for mAbs 2.1 and 9.1 given thatboth 2.1 and 9.1 effectively displaced binding of biotinylated 1109 butshowed only a marginal ability to displace each other. Three mAbs (1E9,7B2, and 8G4) were characterized as partial blockers in the competitiveBJAB binding assay. In the epitope mapping ELISA, these mAbs appeared tobind more peripherally to the central BAFF blocking site given that theyonly partially inhibited the binding of the 11G9, 2.1, and 9.1. Finally,the non-blocking mAb, 12B12, appeared to bind still further away fromthe region of the blocking antibodies given that it could be displacedby only 1E9, a partial blocker.

Mapping studies were also performed to evaluate the binding of V3-1m,B9C11, and P1B8 to mouse BR3. The results demonstrated that while thetwo blocking mAbs (V3-1m and B9C11) were able to cross-compete forbinding to mouse BR3, the non-blocking mAb P1B8 appeared to bind to aseparate epitope.

Example 4 Antagonistic and Agonistic Effects of Anti-BR3 Antibodies on BCell Proliferation

(a) 2.1, 9.1 and 1109 Inhibit Human B Cell Proliferation

B cells were isolated from peripheral blood mononuclear cells bypositive selection using CD19 MACS beads (Miltenyi Biotec). Forproliferation assays, B cells were set up cells at 2×10⁵ c/well inflat-bottom 96-well plate in triplicate. Cells were cultured cells for 5days with anti-IgM (10 mg/ml) (Jackson Immunoresearch), mBAFF (5 μg/ml)and the indicated anti-BR3 antibodies or proteins for 5 days. Antibodiesused were chimeric antibodies in an hIgG1 background and purified fromtissue culture. The cells were then pulsed with 1 mCi/welltritiated-thymidine for the last 6 hours of culture, harvested onto afilter and counted.

(b) V3-1 Inhibits Murine B Cell Proliferation

Splenic B cells were prepared from C57BL/6 mice or from anti-HEL BCRtransgenic mice at the age of 2-4 months, using B cell isolation kitfrom Miltenyi, according to the manufacture's instruction. B cells withmore than 95% purity were consistently obtained. The B cells werecultured in the RPMI-1640 medium, containing 10% heat-inactivated FCS,penicillin/streptomycin, 2 mM L-glutamine and 5×10⁻² μMbeta-Mercaptoethanol.

The purified B cells (10⁵B cells at final volume of 200 μl) werecultured with anti-mouse lgM Ab 5 μg/ml (IgG, F(ab′)₂ fragment) (JacksonImmunoResearch Laboratories) or Hen Egg Lysozyme (Sigma), with orwithout BAFF (2 ng/ml or 10 ng/ml), in the absence or presence ofvarious concentration of anti-BR3 mAbs. Proliferation was measured by³H-thymidine uptake (1 uCi/well) for the last 8 hours of 48 hourstimulation. In some experiments, anti-BR3 mAbs as well as BR3-Fc fusionprotein were pre-boiled for 5 min using PCR machine to inactivate them(controls).

Both B9C11 and V3-1m can inhibit the BAFF costimulatory activity duringanti-IgM mediated primary murine B cell proliferation. Neither B9C11 norV3-1m showed any direct effect on B cell proliferation in the absence orpresence of various doses of anti-IgM antibody (data not shown).Inhibition of proliferation of B cells from anti-HEL BCR transgenic micewith V3-1m and B9C11 (not boiled V3-1m or B9C11) was also observed (datanot shown). Both antibodies are not agonistic in that they do nottrigger normal murine B cells proliferation on their own.

(a) Other Antibodies

Human B cells were isolated from peripheral blood mononuclear cells bypositive selection using CD19 MACS magnetic beads according to themanufacturer's protocol (Miltenyi Biotec, Auburn, Calif.). Cells wereeither used immediately after isolation or were frozen in liquidnitrogen for later use; fresh and frozen cells performed equivalently inthe assay. B cells were cultured at 1×10⁵ cells/well in black 96-wellplates with clear, flat-bottomed wells (PE Biosystems, Foster City,Calif.).

For evaluating antagonistic effects of anti-BR3 antibodies, the cellswere incubated with soluble recombinant BAFF (10 ng/ml) and a F(ab′)2goat anti-human IgM (Fc specific) antibody (4 μg/ml) (JacksonImmunoResearch, West Grove, Pa.) in the presence and absence of variousconcentrations of anti-BR3 antibody ranging from 100 nM to 1.3 μM (15μg/ml-1 ng/ml). B cell proliferation was assessed at day 6 by addingCeiltiter Glo (Promega, Madison, Wis., reconstituted according themanufacturer's instructions) to each assay well. The plates were thenread in a luminometer after incubation for 10 minutes at roomtemperature.

The potential for anti-BR3 antibody agonism to stimulate B cellproliferation was assessed by incubating anti-BR3 antibody (100 nM to1.3 pM) in the presence of the anti-IgM antibody alone (4 μg/ml) or inthe presence of anti-IgM plus a ‘cross-linking’ F(ab′)2 goat anti-humanIgG Fc antibody (Pierce, Rockford, Ill., 30 μg/ml) and in the absence ofBAFF. Proliferation was assessed at day 6 using Ceiltiter Glo asdescribed above.

9.1-RF blocked BAFF-dependent B cell proliferation and does not agonize.2.1-46 stimulated B cell proliferation in the presence of anti-IgM,indicating that it can act as an agonist.

Example 5 Affinity Measurements Using Biacore Materials and Methods

Real-time biospecific interactions were measured by surface plasmonresonance using Pharmacia BIAcore® 3000 (BIAcore AB, Uppsala, Sweden) atroom temperature (Karlsson, R., et al. (1994) Methods 6:97-108; Morton,T. A. and Myszka, D. G. (1998) Methods in Enzymology 295: 268-294).Human BR3 ECD or vBR3-Fc was immobilized to the sensor chip (CM5)through primary amine groups. The carboxymethylated sensor chip surfacematrix was activated by injecting 20 μl of a mixture of 0.025 MN-hydroxysuccinimide and 0.1 M N-ethyl-N′(dimethylaminopropyl)carbodiimide at 5 μl/min. 5-10 μl of 5 μg/ml solution of BR3 ECD orvBR3-Fc in 10 in/VI sodium acetate, pH 4.5, were injected at 5 μl/min.After coupling, unoccupied sites on the chip were blocked by injecting20 μl of 1M ethanolamine, pH 8.5. The running buffer was PBS containing0.05% polysorbate 20. For kinetic measurements, two-fold serialdilutions of anti-BR3 antibodies (6.2-100 nM or 12.5-200 nM) in runningbuffer were injected over the flow cells for 2 minutes at a flow rate of30 μl/min and the bound anti-BR3 antibody was allow to dissociate for 20minutes. The binding surface was regenerated by injecting 20-30 μl of 10mM glycine•HCl (pH 1.5). Flow cell one, which was activated but did nothave BR3 ECD or BR3-Fc immobilized, was used as a reference cell. Therewas no significant non-specific binding of anti-BR3 antibodies to flowcell one. Data were analyzed using a 1:1 binding model using globalfitting. The association and dissociation rate constants were fittedsimultaneously (BIAevaluation software). Similar results were obtainedwhether samples were run in the order of increasing or decreasingconcentrations for selected antibodies tested.

Binding kinetics of anti-BR3 antibodies to BR3 ECD or BR3-Fc weremeasured by BIAcore. BR3 ECD or vBR3-Fc was immobilized on sensor chips,and serial dilutions of antibodies were injected over the flow cells.Alternatively, anti-BR3 antibodies were immobilized on sensor chips, andserial dilutions of BR3 ECD were injected over the flow cells. A highflow rate was used in order to minimize mass transport effects. Resultsof humanized Fab and humanized IgG antibodies compared side by side. Theapparent binding affinities obtained using IgG in solution are higherthan those obtained using Fab in solution, likely due to the avidityeffects since IgG is bivalent.

Example 6 Functional Epitope Mapping

The following assays were used to functionally map the epitopes on BR3important for anti-BR3 antibody binding.

Library Construction for miniBR3 Shotgun Scanning. Libraries displayingepitope-tagged p-miniBR3 on M13 bacteriophage were constructed bysuccessive mutageneses of phagemid pW1205a as previously described(Weiss, G. A., et al., (2000) Proc Natl Acad Sci USA 97:8950-4; Gordon,N. et al., (2003) Biochemistry 42:5977-83). This phagemid encodes apeptide epitope tag (MADPNRFRGKDLGG) fused to the N-terminus of humangrowth hormone followed by M13 gene-8 major coat protein. pW1205a wasused as a template for Kunkel mutagenesis (Kunkel, J. D., et al., (1987)Methods Enzymol 154:367-82) to generate appropriate templates forminiBR3 shotgun library construction. Oligonucleotides replaced thefragment of pW1205a encoding human growth hormone with DNA fragmentsencoding a partial sequence of miniBR3 containing TAA stop codons inplace of the region to be mutated. The two new templates generated,template 1 (encoding residues 34-42) and template 2 (encoding residues17-25), were each used to construct a miniBR3 library as previouslydescribed (Sidhu, H. et al., Methods Enzymol 328:333-63). Each “partialminiBR3” template was used as the template for Kunkel mutagenesis withmutagenic oligonucleotides designed to replace the template stop codonswith the complementary region of miniBR3, while simultaneouslyintroducing mutations at the desired sites. At the sites of mutation,wild-type codons were replaced with the corresponding shotgun alaninecodon (Weiss, supra). Each of these two libraries allowed for mutationsat 11 residues in miniBR3 with no mutated positions in common betweenlibraries. Library 1 encoded shotgun codons at positions 17, 18, 20-23,25, 27, 28, 30, and 33, while library 2 encoded shotgun codons atpositions 26, 29, 31, 34, and 36-42. Each library contained 2×10⁹members, allowing for complete representation of the theoreticaldiversity (>10⁴-fold excess).

Library Sorting and Analysis. Phage from each of the two librariesdescribed above were subjected rounds of binding selection against theneutralizing antibodies 9.1, 2.1, 8G4, 11G9 (functional selection) andV3-1 or an anti-tag antibody (3C8:2 F4, Genentech, Inc.) (displayselection) immobilized on 96-well Nunc Maxisorp immunoplates. Thedisplay selection was included in order to normalize the anti-BR3antibody-binding selection for expression differences between librarymembers. Phage eluted from each target were propagated in E. coliXL1-blue; amplified phage were used for selection against the sametarget as in the previous round. After two rounds of selection, 48individual clones from each library and selection were grown in a96-well format in 400 L of 2YT medium supplemented with carbenicillinand KO7 helper phage. Supernatants from these cultures were useddirectly in phage ELISAs to detect phage-displayed variants of miniBR3capable of binding the antibody target they were selected against toconfirm binding.

Phage ELISA can be performed generally as followed. Maxisorpimmunoplates (96-well) were coated with capture target protein (anti-BR3antibody) for two hours at room temperature (100 μl at 5 μg/ml in 50 mMcarbonate buffer (pH 9.6)). The plates were then blocked for one hourwith 0.2% BSA in phosphate-buffered saline (PBS) and washed eight timeswith PBS, 0.05% Tween 20. Phage particles were serially diluted into BSAblocking buffer and 100 μl was transferred to coated wells. After onehour, plates were washed eight times with PBS, 0.05% Tween 20, incubatedwith 100 μl of 1:3000 horseradish peroxidase/anti-M13 antibody conjugatein BSA blocking buffer for 30 minutes, and then washed eight times withPBS, 0.05% Tween 20 and twice with PBS. Plates were developed using ano-phenylenediamine dihydrochloride/H₂O₂ solution (100 μl), stopped with2.5 M H₂SO₄ (50 μl), and absorbance measured at 492 nm.

All clones tested were found to be positive in their respective ELISAsand were then sequenced as previously described (Weiss, supra).Sequences of acceptable quality were translated and aligned.

Data for BAFF binding and display selection were previously measured(Gordon, supra). Data for anti-BR3 binding and display selection wassimilarly calculated. Generally, the occurrence of the wild-type residue(wt) and each ala mutation (mut) found amound sequenced clones followingtwo rounds of selection for binding to anti-BR3 antibody or anti-tagantibody was tabulated. The occurrence of the wild-type residue wasdivided by that of the mutant to determine a wt/mut ratio for eachmutation at each position (not shown).

F-values were calculated as previously described (Weiss, supra-, Gordon,supra). Generally, a normalized frequency ratio (F) was calculated toquantify the effect of each BR3 mutation on BAFF or anti-BR3antibody-binding while accounting for display efficiencies: i.e.,F=[wt/mutant(BAFF or anti-BR3 antibody selection)] divided by[wt/mutant(display selection)]. Deleterious mutations have ratios >1,while advantageous mutations have ratios <1; boldface indicatesa >10-fold effect. Mutations that showed a greater than 10-fold effect(i.e., F>10 or F<0.1) were considered particularly significant.

TABLE 4 F values Residue 9.1 2.1 8G4 11G9 V3-1 BAFF T17 0.6 0.6 1.5 0.50.5 0.9 P18 0.4 0.5 1.5 0.5 0.8 0.9 C19 V20 0.6 3 2.1 1.1 0.9 1.4 P21 11.9 62 40 0.6 0.5 A22 0.3 3.2 69 45 0.7 0.7 E23 4.8 9.6 11 6.9 2.4 5.4C24 F25 81 49 58 38 21 46 D26 8.7 6.1 6.4 8.5 8.7 17 L27 2.1 0.8 12 1.11.4 9.5 L28 1.5 0.1 2.5 0.4 98 210 V29 0.3 0.5 0.8 1 92 57 R30 10 10 111.7 20 16 H31 0.5 0.6 3.8 2.8 0.1 0.3 C32 V33 10 10 38 24 14 106 A34 1462 41 32 13 28 C35 G36 1.9 14 1.7 1.8 0.7 1.3 L37 0.7 0.1 0.8 0.7 0.75.4 L38 89 0.9 1 0.9 1.4 47 R39 63 0.5 2.2 3.1 0.4 4.1 T40 0.4 0.2 0.50.5 0.6 0.5 P41 7.2 0.7 1.7 1.7 1.6 1.9 R42 2.2 1.8 0.8 0.9 0.9 1.5

The data indicates that 11G9, 9.1, and 2.1 exploit regions of sequencevariation between human and murine BR3 (Table 4). The functional epitopefor V3-1 mimics the functional epitope for BAFF that is highly conservedbetween human and murine BR3. 11G9, 2.1, 9.1, and V3-1 antibodies do notrequire BR3 glycosylation for binding. The functional epitope for the9.1 antibody includes L38 and R39. The functional epitope for 2.1includes G36. The functional epitope for V3-1 includes L28 and L29. Thefunctional epitope for 11G9 includes P21 and A22. Alanine scanningmutation of residues A34, F25 and V33 also disrupted 9.1, 2.1, 11G9, andV3-1 binding to BR3 in this assay, which residues may be important formaintaining the structural integrity of BR3 in the phage.

Example 7 Antibody Dependent Cellular Cytotoxicity

Anti-BR3 chimeric monoclonal antibodies were assayed for their abilityto mediate Natural-Killer cell (NK cell) lysis of BJAB cells (ADCCactivity), a CD20 expressing Burkitt's lymphoma B-cell line, essentiallyas described (Shields et al., J. Biol. Chem. 276:6591-6604 (2001)) usinga lactate dehydrogenase (LDH) readout. NK cells were prepared from 100mL of heparinized blood from normal human donors using the RosetteSep®Human NK Cell Enrichment Cocktail (StemCell Technologies, Vancouver, B.C.) according to the manufacturer's protocol. The blood was diluted withan equal volume of phosphate buffered saline, layered over 15 mL ofFicoll-Paque™ (Amersham Biosciences, Uppsala, Sweden), and centrifugedfor 20 min at 1450 RPM. White cells at the interface between layers weredispensed to 4 clean 50-mL tubes, which were filled with RPMI mediumcontaining 15% fetal calf serum. Tubes were centrifuged for 5 min at1450 RPM and the supernatant discarded. NK cells were diluted in assaymedium (F12/DMEM 50:50 without glycine, 1 mM HEPES buffer pH 7.2,Penicillin/Streptomycin (100 units/mL; Gibco), glutamine, and 1%heat-inactivated fetal bovine serum) to 2×10⁶ cells/mL.

Serial dilutions of antibody (0.05 mL) in assay medium were added to a96-well round-bottom tissue culture plate. BJAB cells were diluted inassay buffer to a concentration of 4×10⁵/mL. BJAB cells (0.05 mL perwell) were mixed with diluted antibody in the 96-well plate andincubated for 30 min at room temperature to allow binding of antibody toBR3 (opsonization).

The ADCC reaction was initiated by adding 0.05 mL of NK cells to eachwell. In control wells, 2% Triton X-100 was added. The plate was thenincubated for 4 h at 37° C. Levels of LDH released were measured using acytotoxicity (LDH) detection kit (Kit# 1644793, Roche Diagnostics,Indianapolis, Ind.) following the manufacturers instructions. 0.1 mL ofLDH developer was added to each well, followed by mixing for 10s. Theplate was then covered with aluminum foil and incubated in the dark atroom temperature for 15 min. Optical density at 490 nm was then read andused to calculate % lysis by dividing by the total LDH measured incontrol wells. Lysis was plotted as a function of antibodyconcentration, and a 4-parameter curve fit (KaleidaGraph) was used todetermine EC₅₀ concentrations.

All humanized anti-BR3 antibodies were strongly active in directing NKcell mediated lysis of BJAB cells (human Burkitt's Lymphoma) withrelative potencies less than 1 nM. Similar assays were carried out withRamos (human Burkitt's lymphoma) and WIL2s cells (human B-cell lymphoma)instead of BJAB cells. ADCC killing of Ramos and WIL2s cells wasobserved with anti-BR3 antibodies. An anti-Her2 antibody (4D5) was usedas a negative control. In general, antibodies with higher affinity forBR3 were more potent in antibody-dependent cell-killing assays.

Example 8 Depletion of B Cells with BR3-Fc or Anti-BR3 Antibodies

The ability of anti-BR3 antibodies to deplete B cells was compared withBR3-Fc. Six week old BALB/c mice were treated interperitoneally at day 0with 500 μg control (mouse IgG2a), mouse BR3-Fc or anti-BR3 (V3-1)antibodies. Mice from each group were sacrificed at day 1, 3, 7 and 15.Flowcytometry analysis of B cells in the blood, lymph nodes and spleenat day 7 of treatment was conducted. The blood, lymph nodes and spleenshow fewer B cells (CD21+CD23+ and CD21highCD23low) in V3-1 treated micethan in BR3-Fc and control treated animals. BR3-Fc treatment haspreviously been shown to significantly reduce the number of B cellscompared with control Fc treated animals.

In another experiment under similar conditions, FACS analysis of blood,lymph nodes and spleen generally showed fewer B cells (CD21+CD23+ andCD21highCD23low) in V3-1 treated mice than in BR3-Fc and control treatedanimals. BR3-Fc significantly reduced the number of B cells comparedwith control animals particularly at later time points. The absolutenumber of B cells contained in 1 ml of blood; the % of B cells in lymphnodes and the absolute numbers of follicular (FO—CD21+CD23+) or marginalzone (MZ—CD21high CD23low) in the spleen at days 1, 3, 7 and 15 weredetected. Data were expressed as the mean+/−standard error (n=4).

In another experiment under similar conditions, FACS analysis ofplasmablasts in the spleen (top row—IgM+Syn+) and germinal center cells(middle row—B220+CD38low) show that anti-BR3 antibodies (V3-1) candeplete some plasmablasts and germinal center cells. BR3-Fcsignificantly reduced the number of plasmablasts compared with controlanimals.

The data showed that a greater extent of B cell depletion was observedafter treatment with anti-BR3 antibodies than with BR3-Fc, which fusionprotein blocks BAFF binding to BR3 but does not have ADCC function.

Example 9 Fc-Dependent Cell Killing and BAFF Blockade For Maximal B CellReduction

BALB/c mice were treated with a single dose 10 mg/kg of anti-BR3antibody (mV3-1), mV3-1 with D265A/N297A mutations, a non-BAFF blockinganti-BR3 antibody PIH11 or BR3-Fc. B cells from spleen or peripheralblood were analyzed by flowcytometry at day 6 post treatment. Theabsolute numbers of peripheral blood B cells (B220+) and splenicfollicular B cells (CD21+CD23+) after treatment are detected.

The D265A/N297A Fc mutation abolished binding of FcγRIII in vitro. Theresults indicate that although both the non-blocking antibody, theanti-BR3 antibodies with defective Fcgamma receptor-binding, and BR3-Fccan reduce B cell populations, the anti-BR3 antibody having bothFc-dependent cell killing activity and BAFF-blocking activity can be amuch more potent B cell reducing/depleting agent. This is due tocombining both activities, antibody dependent cell cytotoxicity (ADCC)and B cell survival blockade, into one molecule.

Example 10 Fcγ Receptor Binding

Human FcγRs (also referred to as hFcgR below) lacking theirtransmembrane and intracellular domains and comprising His-taggedglutathione S transferase (GST) sequences at their C-terminus wereprepared as described previously (Shields, R. L. et al., (2001) JBC276:6591-6604).

MaxiSorp 96-well microwell plates (Nunc, Roskilde, Denmark) were coatedwith 2 μg/ml anti-GST (clone 8E2.1.1, Genentech), at 100 μl/well in 50mM carbonate buffer, pH 9.6, at 4° C. overnight. Plates were washed withPBS containing 0.05% polysorbate, pH 7.4 (wash buffer) and blocked withPBS containing 0.5% BSA, pH 7.4, at 150 μl/well. After an hourincubation at room temperature, plates were washed with wash buffer.Human Fcγ receptor was added to the plates at 0.25 μg/ml, 100 μl/well,in PBS containing 0.5% BSA, 0.05% polysorbate 20, pH 7.4 (assay buffer).The plates were incubated for one hour and washed with wash buffer. Forlow affinity Fcγ receptors IIa, IIb, III (F158) and high affinity III(V158), antibodies were incubated with goat F(ab′)₂ anti-κ (Cappel, ICNPharmaceuticals, Inc., Aurora, Ohio) or anti-λ. (BioSource, Camarillo,Calif.) antibody at a 1:2 (w/w) ratio for 1 hour to form antibodycomplexes. Eleven twofold serial dilutions of complexed IgG antibodies(1.17-50000 ng/ml in threefold serial dilution) in assay buffer wereadded to the plates. For the high affinity FcγRI, eleven twofold serialdilutions of uncomplexed IgG antibodies (0.017-1000 ng/ml in threefoldserial dilution) in assay buffer were added to the plates. After atwo-hour incubation, plates were washed with wash buffer. Bound IgG wasdetected by adding peroxidase labeled goat F(ab′)₂ anti-human IgGF(ab′)₂ (Jackson ImmunoResearch, West Grove, Pa.) at 100 μl/well inassay buffer. After a one-hour incubation, plates were washed with washbuffer and the substrate 3,3′,5,5′-tetramethyl benzidine (TMB)(Kirkegaard & Perry Laboratories) was added at 100 μl/well. The reactionwas stopped by adding 1 M phosphoric acid at 100 μl/well. Absorbance wasread at 450 nm on a multiskan Ascent reader (Thermo Labsystems,Helsinki, Finland).

The absorbance at the midpoint of the standard curve (mid-OD vs. ng/ml)was calculated. The corresponding concentrations of standard and samplesat this mid-OD were determined from the titration curves using afour-parameter nonlinear regression curve-fitting program (KaleidaGraph,Synergy software, Reading, Pa.). The relative activity was calculated bydividing the mid-OD concentration of standard by that of sample. TheHerceptin® Ab has previously been shown to bind Fcγ Receptors and wasused as a positive control here.

For all FcγR, binding values reported are the binding of each 9.1-RFvariant relative to 9.1RF, taken as(A_(450 nm(variant))/A_(450 nm(9/1RF))) at 0.33 or 1 μg/ml for FcγRIIand FcγRIIIA and 2 μg/ml for FcγRI. A value greater than 1 denotesbinding of the variant was improved compared with 9.1RF, whereas a ratioless than 1 denotes reduced binding compared with 9.1RF. ThehFcγRIII(F158) and hFcγRIII(V158) refer to hFcγRIII isotypes havinglower affinity and higher affinity for human IgG, respectively.

The 9.1 anti-BR3 antibodies bind FcγRs similarly and should promoteADCC.

TABLE 5 Relative binding to Fcγ receptors. hFcgRIII hFcgRIII AntibodyhFcgRI hFcgRIIa hFcgRIIb (F158) (V158) Herceptin ® 1.02 0.54 0.62 0.510.80 Ab 9.1-RF 1.00 1.00 1.00 1.00 1.00 9.1-RF N434A 0.97 0.66 0.45 0.420.58 9.1-RF N434W 1.00 0.64 0.40 0.24 0.51

Example 11 Anti-BR3 Antibodies with Altered ADCC Activity

(a) FcgammaR Binding

By site-directed mutagenesis, the Fc region of the 9.1RF antibodies weremutated as follows: S298A/K326A/E333A/K334A (“9.1(5)”),S298A/E333A/K334A (“9.1(6)”), 239D/332E (“9.1(7)”) 239D/298A/332E(“9.1(8)”), 239D/268D/298A/332E (“9.1(9)”), 239D/268D/298A/326A/332E(“9.1(10)”), 239D/268D/283L/298A/332E (“9.1(11)”) or239D/268D/283L/298A/326A/332E (“9.1(12)”). Additionally, bysite-directed mutagenesis, the Fc region of the V3-46s antibody wasmutated as follows: S298A/K326A/E333A/K334A (“V3(5)”), 239D/332E(“V3(7)”) or 239D/298A/332E (“V3(8)”). Oligonucleotides specifying theamino acid substitutions were chemically synthesized and used foroligonucleotide-directed mutagenesis of plasmid encoding 9.1RF accordingto the protocol of Kunkel et al. (Methods in Enzymology (1987) 154,367-382). Variant sequences were confirmed by dideoxynucleotide-basedsequencing. Plasmid DNA was purified from 1 L cultures (2YT mediacontaining 50 μg/mL carbenecillin) of E. coli XL-1 Blue (Stratagene,Inc.), transformed with the relevant plasmid and grown at 37° C. withshaking at 200 RPM, by using the gigaprep protocol described by Qiagen,Inc. Proteins were expressed by using the purified plasmid DNA fortransient transfection of CHO cells or 293 cells. Antibodies werepurified from 1 L of culture supernatant by chromatography on ProteinA-Sepharose followed by cation exhange chromatography on SP-Sepharose.The identity of the purified protein was confirmed by SDS-PAGE and aminoterminal sequencing. All of the purified antibodies produced ahomogeneous peak upon analytical gel filtration chromatography, with amolar mass of 150,000±5000 calculated from static light scattering data,and less than 3% aggregate content. Analysis of N-linkedoligosaccharides by MALDI-TOF indicated a carbohydrate compositiontypical of recombinant antibodies (Table 6).

TABLE 6 MALDI-tof analysis of released N-linked oligosaccharides from9.1 Fc variants. Oligosaccharide Oligosaccharide OligosaccharideOligosaccharide Oligosaccharide Oligosaccharide area % area % area %area % area % area % Variant Man5 1100 2000 2100 2110 2120 9.1(5) 1 2 776 13 1 9.1(6) 2 3 6 77 11 1 9.1(7) 1 1 2 64 26 4 9.1(8) 1 1 2 54 35 7

Binding of the variant antibodies to Fcγ receptors was evaluated usingan ELISA-based assay. The extracellular domains of human Fcγ receptorsI, IIa, IIb, IIIa(F158), IIIa(V158) and mouse Fcγ receptors I, II, andIII, were expressed as His-tagged, GST fusion proteins in CHO cells andpurified as described in Shields et al. (J. Biol. Chem. 276:6591-6604(2001)). For the ELISA assay, the fusion proteins were captured on wellsof microtiter plates that had been coated with an anti-GST antibody.Dilutions of the variant antibodies were added and allowed to bindfollowed by washing of the wells to remove unbound antibody. For theweaker binding-antibodies the samples were complexed with a Fab′2fragment of an anti-hu γ-chain antibody prior to addition of the samplesto the wells. Bound antibody was detected with an HRP-coupled, Fab′2fragment of a goat anti-huFab′2 antibody. Binding curves were evaluatedby using a 4-parameter equation to calculate the EC_(so) value, theconcentration of antibody that gives 50% of the signal observed atsaturation. Herceptin® was used as the control antibody in these assaysand the fold improvement in binding was calculated from the ratio of theEC₅₀ values (EC₅₀herceptin/EC₅₀sample).

Table 7 shows that the variants had insignificant changes in affinityfor human FcγRI. Only 9.1 (7) and 9.1(8) had increased affinity forhuman FcγRIIb with 9.1(7) being the tightest binder. 9.1(7) and 9.1(8)had increased affinity for all three mouse Fcγ receptors whereas theaffinity for 9.1(5) and 9.1(6) was unchanged.

TABLE 7 Human Human Human Human Human Mouse Mouse Mouse Antibody IIIaIIIa I IIa IIb (F158) (V158) I II III WT 1.0 2.3 0.7 2.3 1.6 1.2 0.7 0.89.1(5) 0.6 0.2 0.7 25 9.2 1.9 1.3 1.2 9.1(6) 0.7 0.2 0.4 18 6.9 2.5 0.40.6 9.1(7) 0.7 5.3 17 110 18 62 19 9.9 9.1(8) 0.7 0.9 3.5 160 34 24 7.64.0

All of the anti-BR3 variants in Table 7 had increased affinity for boththe F158 and V158 allotypes of human FcγRIIIa, with 9.1(8) having thegreatest affinity for the receptor. FIG. 3 shows the fold change inbinding of V3(8), V3(7), V3(5) and V3-46s to mouse FcγR (I, II, III, IV)or human FcγR (I, III-F158) relative to a control antibody (theHerceptin® antibody). Large Increases in binding affinity to mFcγRI andhRIII-F158 were observed for V3(7) and V3(8).

(b) ADCC Activity

The anti-BR3 antibodies were assayed for their ability to mediateNatural-Killer cell (NK cell) lysis of BJAB cells (ADCC activity), a BR3and CD20 expressing Burkitt's lymphoma B-cell line, essentially asdescribed (Shields et al., J. Biol. Chem. 276:6591-6604 (2001)) using alactate dehydrogenase (LDH) readout. NK cells isolated from donorsheterozygous for the F/V 158 allotype of CD16 were used in the assay atan effector:target ratio of 5:1. NK cells were prepared from 100 mL ofheparinized blood using the RosetteSep® Human NK Cell EnrichmentCocktail (StemCell Technologies, Vancouver, B. C.) according to themanufacturer's protocol. The blood was diluted with an equal volume ofphosphate buffered saline, layered over 15 mL of Ficoll-Paque™ (AmershamBiosciences, Uppsala, Sweden), and centrifuged for 20 min at 1450 RPM.White cells at the interface between layers were dispensed to 4 clean50-mL tubes, which were filled with RPMI medium containing 15% fetalcalf serum. Tubes were centrifuged for 5 min at 1450 RPM and thesupernatant discarded. NK cells were diluted in assay medium (F12/DMEM50:50 without glycine, 1 mM HEPES buffer pH 7.2, Penicillin/Streptomycin(100 units/mL; Gibco), glutamine, and 1% heat-inactivated fetal bovineserum) to 2×10⁶ cells/mL.

Serial dilutions of antibody (0.05 mL) in assay medium were added to a96-well round-bottom tissue culture plate. BJAB cells were diluted inassay buffer to a concentration of 4×10⁵/mL. BJAB cells (0.05 mL perwell) were mixed with diluted antibody in the 96-well plate andincubated for 30 min at room temperature to allow binding of antibody toBR3 (opsonization).

The ADCC reaction was initiated by adding 0.05 mL of NK cells to eachwell. In control wells, 2% Triton X-100 was added. The plate was thenincubated for 4 h at 37° C. Levels of LDH released were measured using acytotoxicity (LDH) detection kit (Kit#1644793, Roche Diagnostics,Indianapolis, Ind.) following the manufacturers instructions. 0.1 mL ofLDH developer was added to each well, followed by mixing for 10s. Theplate was then covered with aluminum foil and incubated in the dark atroom temperature for 15 min. Optical density at 490 nm was then read andused to calculate % lysis by dividing by the total LDH measured incontrol wells. Lysis was plotted as a function of antibodyconcentration, and a 4-parameter curve fit (KaleidaGraph) was used todetermine EC50 concentrations.

FIG. 1 shows that the anti-BR3 variants were active in the ADCC assaygiving EC₅₀ values less than 1 nM (% killing vs antibody concentration).The Fc substitutions led to an increase in potency relative to 9.1RF(data not shown) by the lowering of the EC₅₀ and increase in the maximal% killing. The S298A/K326A/E333A/K334A mutant had a 3 fold higher ADCCactivity in this assay relative to 9.1RF (relative EC₅₀ values). TheS298A/E333A/1C334A mutant had a 2.8 fold higher ADCC activity in thisassay relative to 9.1RF (relative EC₅₀ values). In this comparison,9.1(8) had the highest activity, about a 12-fold lowering of the EC50relative to 9.1RF, and both 9.1(7) and 9.1(8) had higher activity thanan anti-CD20 antibody, Rituximab® antibody, on this cell line.

Additional experiments also showed that 9.1(8) was the Fc variant havingthe most potent ADCC activity on WIL2-S and BJAB cell lines (FIGS. 2Aand 2B). Table 8 is a table listing the relative fold increase in ADCCactivity of the Fc variants compared to 9.1RF. Studies showed that9.1(5) and 9.1(8) were not agonistic for B cell proliferation.

TABLE 8 ABR var. 239 268 283 298 326 332 333 334 Expression ADCCWT(9.1RF) S H E S K I E K 9.1(5) — — — A A — A A CHO 3 9.1(6) — — — A —— A A CHO 3 9.1(7) D — — — — E — — CHO 7 9.1(8) D — — A — E — — CHO 129.1(9) D D — A — E — — 293 10  9.1(10) D D — A A E — — 293 10  9.1(11) DD L A — E — — 293 ND  9.1(12) D D L A A E — — 293 ND

The V3 Fc variants displayed variable, low to substantial increases inADCC activity (FIGS. 4A and 4B). In these particular experiments, thelimits of dynamic range may have been reached and variability in donornatural killer cells may have contributed to the results.

(c) B Cell Depletion with Fc Variants

hCD20 Tg+/+mCD16−/− hCD16 Tg+/+ mice were intravenously treated with 125μg or 12.5 μg V3-46s, V3(5) or V3(8) variants at 125 or 125 μg of ahIgG1 negative control (e.g., Herceptin® antibody) as described in FIG.5. B cells from the blood and spleen of the mice were assayed by FACS onday 7 after administration. FIG. 6 shows that the lower dose of Fcvariant (12.5 μg) resulted in an increased depletion of blood B cellsrelative to wild-type. At the timepoint assayed, variants V3(3) andV3(8) showed similar results. No significant change for spleen B cellswas observed at day 7 (data not shown).

Additional data indicates that anti-BR3 antibody variants of thisinvention can have potents ADCC activity but have little or noneutrophil killing activity compared to a B cell depleter such as ananti-CD20 antibody (data not shown). These results indicate thatanti-BR3 antibody variants of this invention may have less toxicity thatan anti-CD20 antibody such as V511 (data not shown).

Example 12 Underfucosylated Antibody Compositions (a) Conversion of anExisting Cell Line to an Underfucosylated Cell Line.

In order to achieve high yields of non-fucosylated antibodies inmammalian cells, a RNAi approach was employed to knock down theexpression of FUT8 gene. A plasmid was used to produce short hairpinsiRNA consisting of 19 nt (nucleotide) sense siRNA sequence specific tothe gene of FUT8, linked to its reverse complementary antisense siRNAsequence by a short spacer (9 nt hairpin loop), followed by 5-6 U′ s at3′ end.

Four different RNAi probes were designed (probe #1-4) to target thedifferent regions based on the available CHO FUT8 DNA sequence (FIGS. 8and 9).

To test the efficacy of these RNAi probes, a FLAG-tagged FUT8 fusionprotein was constructed using the available CHO FUT8 DNA sequence(Genbank accession no. P_AAC63891). RT-PCR was performed with FUT8primers and the resulting PCR fragment was fused with 5′ FLAG tagsequence. The tagged FUT8 fragment was cloned into an expression vector.The RNAi probe plasmid and flag-tagged FUT8 plasmid were cotransfectedinto CHO cells. Cell lysate was extracted 24 hours after transfectionand the FUT8 fusion protein level was analyzed by anti-flag M2 antibodyby immuno blotting. In the presence of RNAi probes, the fusion proteinexpression was significantly inhibited in four out of the five cases(data not shown).

Probe#2 (RNAi2) and #4 (RNAi4) showed the best inhibitory effect andwere chosen for further evaluation.

Probes 2 and 4 were transfected into a CHO cell line expressing aantibody described herein. The expressed anti-BR3 antibody was purifiedby a protein A column and submitted for MALDI-TOF fucose content andFcγR binding assay (described below). The RNAi transiently transfectedcells produced about approximately 50%-52% nonfucosylated 9.1RF(“a-9.1RF”) and nonfucosylated 9.1(5) (“a-9.1(5)”) antibody as shown inFIG. 10. In contrast, the 9.1 variant cell lines not transfected withRNAi plasmids had 2-7% nonfucosylated antibodies (see 2000 m/z column).The a-9.1RF antibody pool and a-9.1(5) antibody pool having 50-52%nonfucosylation=showed an approximate 3-10 fold increase in ADCCactivity (FIG. 11) and an increase in binding affinity towards FcγRIII(F158 allele) and FcγRIII (V158 allele) (FIGS. 12A and 12B). No effectwas seen with hFcγR1, hFcγRIIa and hFcγRIIb (FIGS. 12A and 12B).

To confirm that the RNAi transfected cells do have less FUT8 RNAexpression, a Northern blot can be performed using RNA samples extractedfrom the transfected cells 24 hours after transfection. Total RNA fromcells containing a control plasmid (random mouse DNA sequence, nohomology to any known mouse proteins) and 2 RNAi plasmids can bepurified and hybridized with a 300 by probe. The knock down ofendogenous a 1,6-fucosyltransferase RNA can be further confirmed byquantitative PCR (data not shown).

(b) Generation of Stable Cell Line with Simultaneous Knockdown ofFucosylation Level

Materials and Methods

Cell Culture and Transfection

Chinese Hamster Ovary (CHO) cells can be grown in growth medium with 5%FBS (fetal bovine serum) and 1×GHT (glycine, hypoxanthine, andthymidine) at 37° C. For transient transfection, DMRIE-C transfectionreagent (Invitrogen) can be used. For stable transfection, Lipofectamine2000 (Invitrogen) can be used. The cells can be transfected with aplasmid having the configuration of FIG. 8. Alternatively, a stablecells lines having a FUT8 knock-out came be made (e.g., method describedin Yamane-Ohnuki, et al., (2004) Biotechnol. Bioeng. 87:614-622).

Selection

After the transfection, cells can be centrifuged to collect the pellet.The pellet can be resuspended in medium containing 25 nM methotrexate(MTX). Medium can be changed every 3 to 4 days. About 2 weeks aftertransfection, individual clones can be picked and grown in 96-wellplates. Usually it takes about 1 week for cells to grow confluent in a96-well plate.

ELISA Assay

When cells are confluent, the growth medium can be removed and theproduction medium can be added into each well. The day after adding theproduction medium, the plate can be incubated at 33° C. for 5-6 daysbefore the ELISA assay. Typically an ELISA is performed with serialdilutions.

RNA Analysis

Total RNA can be purified with Qiagen's RNA purification kit andquantified by Taqman with gene specific primers and probes.

Matrix-Assisted Laser Desorption/Ionization Time-of-flight (MALDI-TOF)Mass Spectral Analysis of Asparagine-Linked Oligosaccharides:

Methods for analyzing the oligosaccharides by MALDI-TOF were conductedgenerally as follows: N-linked oligosaccharides were released fromrecombinant glycoproteins using peptide-N-glycosidase-F (PNGase F)procedure of Papac et al., Glycobiology 8, 445-454 (1998). Briefly, thewells of a 96 well PVDF-lined microtitre plate (Millipore, Bedford,Mass.) were conditioned with 100 μl methanol that was drawn through thePDVF membranes by applying vacuum to the Millipore Multiscreen vacuummanifold. The conditioned PVDF membranes were washed with 3×250 μlwater. Between all wash steps the wells were drained completely byapplying gentle vacuum to the manifold. The membranes were washed withreduction and carboxymethylation buffer (RCM) consisting of 6 Mguanidine hydrochloride, 360 mM Tris, 2 mM EDTA, pH 8.6. Glycoproteinsamples (50 μg) were applied to individual wells, again drawn throughthe PVDF membranes by gentle vacuum and the wells were washed with 2×50μl of RCM buffer. The immobilized samples were reduced by adding 50 μlof a 0.1 M dithiothreitol (DTT) solution to each well and incubating themicrotitre plate at 37° C. for 1 hr. DTT was removed by vacuum and thewells were washed 4×250 μl water. Cysteine residues werecarboxylmethylated by the addition of 50 μl of a 0.1 M iodoacetic acid(IAA) solution which was freshly prepared in 1 M NaOH and diluted to 0.1M with RCM buffer. Carboxymethylation was accomplished by incubation for30 min in the dark at ambient temperature. Vacuum was applied to theplate to remove the IAA solution and the wells were washed with 4×250 μlpurified water. The PVDF membranes were blocked by the addition of 100μl of 1% PVP360 (polyvinylpyrrolidine 360,000 MW) (Sigma) solution andincubation for 30 minutes at ambient temperature. The PVP-360 solutionwas removed by gentle vacuum and the wells were washed 4×250 μl water.PNGase F (New England Biolabs, Beverly, Mass.) at 25 μl of a 25 Unit/mlsolution in 10 mM Tris acetate, pH 8.3, was added to each well and thedigest proceeded for 3 hr at 37° C. After digestion, the samples weretransferred to 500 μl Eppendorf tubes and 2.5 μl of a 1.5 M acetic acidsolution was added to each sample. The acidified samples were incubatedfor 2 hr at ambient temperature to convert the oligosaccharides from theglycosylamine to the hydroxyl form. Prior to MALDI-TOF mass spectralanalysis, the released oligosaccharides were desalted using a 0.7-ml bedof cation exchange resin (AG50W-X8 resin in the hydrogen form) (Bio-Rad,Hercules, Calif.) slurried packed into compact reaction tubes (USBiochemical, Cleveland, Ohio).

For MALDI-TOF mass spectral analysis of the samples in the positivemode, the desalted oligosaccharides (0.5 μl aliquots) were applied tothe stainless target with 0.5 μl of the 2,5 dihydroxybenzoic acid matrix(sDHB) that was prepared by dissolving 2 mg 2,5 dihydroxybenzoic acidwith 0.1 mg of 5-methoxyslicylic acid in 1 ml of 1 mM NaCl in 25%aqueous ethanol. The sample/matrix mixture was dried by vacuum. Thesample/matrix mixture was vacuum dried and then allowed to absorbatmospheric moisture prior to analysis. Released oligosaccharides wereanalyzed by MALDI-TOF on a PerSeptive BioSystems Voyager-ELITE massspectrometer. The mass spectrometer was operated in the positive mode at20 kV with the linear configuration and utilizing delayed extraction.Data were acquired using a laser power of approximately 1100 and in thedata summation mode (240 scans) to improve the signal to noise. Theinstrument was calibrated with a mixture of standard oligosaccharidesand the data was smoothed using a 19 point Savitsky-Golay algorithmbefore the masses were assigned. Integration of the mass spectral datawas achieved using Caesar 7.2 data analysis software package (SciBridgeSoftware).

Example 13 BJAB B Cell Binding Assay Using Afucosylated HU9.1RF IgG1Antibody

THE BJAB cell binding assay used to assay the afucosylated anti-BR3antibody Hu9.1RF IgG1 (comprising the VH and VL sequence of SEQ ID NOs:35 and 21, respectively (see Table 2)), was essentially as described inExample 2 with the following minor modifications. BJAB cells werecultured in RPMI media supplemented with 10% fetal bovine serum (FBS,Sigma, St. Louis, Mo.), penicillin (100 U/ml, Gibco-Invitrogen,Carlsbad, Calif.), streptomycin (100 μg/ml, Gibco), and L-glutamine (10mM).

For binding assays, cells were washed with cold assay buffer (phosphatebuffered saline [PBS], pH 7.4) containing 1% FBS and 0.5% bovine serumalbumin [BSA]). The cell density was adjusted to 1.25×10⁶/ml, and 200 μlof cell suspension was aliquotted into the wells of 96 well round bottompolystyrene plates (NUNC, Neptune, N.J.; 250,000 cells/well). The platescontaining the cells were centrifuged at 1500 rpm for 5 minutes at 4′C,and the supernatant was carefully aspirated away from the cell pellets.Direct and competitive binding assays were performed as follows. For thedirect binding assay, antibody samples (Hu9.1RF IgG1 and afucosylatedHu9.1RF IgG1) were serially diluted in cold assay buffer toconcentrations ranging between 16.7-0.001 nM. Samples (100 μl) wereadded to the pelleted cells and the plates were shaken briefly on aplate shaker to loosen the cell pellets. The remainder of the assay wasexactly as described in Example 2.

In the competitive binding assay, anti-BR3 mAbs compete with FLAG-taggedsynthetic murine BAFF (produced at Genentech) for binding to cellsurface BR3. The anti-BR3 antibodies (Hu9.1RF IgG1 and afucosylatedHu9.1RF IgG1) were serially diluted and combined with an equal volume ofFLAG-BAFF to give final concentrations of 50-0.02 nM mAb and 25 nMFLAG-BAFF. The diluted samples were added to the pelleted BJAB cells in96 well plates as described above. After a 45 min incubation on ice, thecells were washed three times with 200 μl cold assay buffer, andanti-FLAG-HRP antibody (Sigma) diluted 1/20,000 in assay buffer wasadded (100 μl/well). The plates were incubated for a final 45 minutes onice. After three washes with cold assay buffer, color was developedusing TMB, the reaction was stopped with H₃PO₄, and the plates were readas described above.

The results of the BJAB binding assays demonstrated that afucosylatedHu9.1RF IgG1 bound to BR3 at least as well as the fucosylated controlversion of the antibody (FIG. 13A). In the direct binding assay, theEC₅₀ for afucosylated Hu9.1RF IgG1 was 0.49 nM as compared to 0.92 nMfor control Hu9.1RF IgG1. Moreover, in the competitive binding assay,afucosylated Hu9.1RF IgG1 displaced BAFF with an IC₅₀ of 3.6 nM ascompared to 8.8 nM for the fucosylated form of the antibody (FIG. 13B).

These experiments demonstrate that the afucosylated form of Hu9.1RF IgG1behaved essentially identically to control Hu9.1RF IgG1 in terms of cellsurface BR3 binding and blockade of BAFF binding.

Example 14 Antagonistic and Agonistic Effects of Afucosylated HU9.1RFIgG1 Antibody

The B cell proliferation assays were performed essentially as describedin Example 4, above with the following modifications. Human B cellsisolated from peripheral blood mononuclear cells were either usedimmediately after isolation or were frozen in liquid nitrogen for lateruse; fresh and frozen cells performed equivalently in the assay. B cellswere cultured at 1×10⁵ cells/well in black 96-well plates with clear,flat-bottomed wells (PE Biosystems, Foster City, Calif.). For evaluatingantagonistic effects of antibodies, the cells were incubated withFLAG-BAFF (20 ng/ml) and a F(ab′)₂ goat anti-human IgM (Fc specific)antibody (4 μg/ml) (Jackson ImmunoResearch) in the presence and absenceof various concentrations of anti-BR3 antibody ranging from 10 nM to0.13 pM (1.5 μg/ml-0.02 ng/ml). B cell proliferation was assayed asdescribed in Example 4. The potential for anti-BR3 antibody agonism wasassessed by incubating the anti-BR3 antibody (10 nM to 0.13 pM) in thepresence of the anti-IgM antibody alone (4 μg/ml). Proliferation wasassessed at day 6 using Celltiter Glo as described above. Hu9.1RF IgG1with an N434W mutation was used as an agonistic positive control andHerceptin was used as a human IgG1 isotype negative control.

In the primary B cell proliferation assay, afucosylated Hu9.1RF IgG1(comprising the VH and VL sequence of SEQ ID NOs: 35 and 21,respectively (see Table 2)), inhibited B cell proliferation stimulatedby BAFF and anti-IgM essentially identically to control Hu9.1RF IgG1(FIG. 14). The control antibodies (Hu9.1RF IgG1 with an N434W mutationand the IgG1 isotype control) had no effect on BAFF/anti-IgM stimulatedproliferation as expected. In order to assess possible agonistic effectsof the antibodies, B cells were stimulated with anti-IgM alone. Theresults showed that neither the control nor the afucosylated Hu9.1RFIgG1 antibodies stimulated B cell proliferation above the level observedwith anti-IgM alone (FIG. 15). The agonistic positive control antibody(Hu9.1RF IgG1 with an N434W mutation) and isotype negative control bothbehaved as expected, with Hu9.1RF IgG1 with an N434W mutation causing adose-dependent increase in proliferation in the presence of anti-IgMalone and the IgG1 control having no effect.

These experiments demonstrate that the afucosylated molecule was equallypotent to control Hu9.1RF IgG1 in terms of inhibiting BAFF-stimulatedprimary B cell proliferation and had no evident agonistic effects.

Example 15 Affinity Measurements for Afucosylated Hu9.1RF IgG1 Antibody

Real-time biospecific interactions for the anti-BR3 antibodies,including the afucosylated Hu9.1RF IgG1 antibody, were measured bysurface plasmon resonance using BIAcore® 3000 (GE Healthcare,Piscataway, N.J.) at room temperature (Karlsson et al., Methods 6,97-108 (1994); Morton & Myszka, Methods in Enzymology, 295, 268-294(1998)).

Two formats were used to assess the binding affinities of the anti-BR3antibodies to BR3 extra cellular domain (BR3 ECD). The first format wassimilar to the format described in Example 5, above with the followingmodifications. Human BR3 ECD (52132-5P) was immobilized to a sensor chip(CM5) through primary amine groups and the sensor chip was activated asdescribed above. A total of 70 ml (sequential injections of 5, 15, 30,10, and 10 ml) of 5 mg/ml solution of BR3 ECD in 10 mM sodium acetate,pH 4.5, was injected at 5 ml/min. For kinetic measurements, two-foldserial dilutions of the anti-BR3 antibodies (1.56-200 nM) in runningbuffer were injected over the flow cells for 2 minutes at a flow rate of30 ml/min. The bound anti-BR3 antibodies were allowed to dissociate for20 minutes before the binding surface was regenerated by injecting 20 mlof 10 mM glycine HCl (pH 1.5). Flow cell one was used as a referencecell as described above. Data were analyzed using the 1:1 Langmuirbinding model, as described above, and using the bivalent analyte model.

For the second format, anti-BR3 antibodies Hu9.1RF IgG1 and theafucosylated Hu9.1RF IgG1 were separately immobilized to individual flowcells on a sensor chip (CM5) through primary amine groups. Theafucosylated Hu9.1RF IgG1 (Hu9.1RF IgG1-AF) for use in the experimentswas generated in a host cell lacking a fucosyltransferase enzyme. TheHu9.1RF IgG1-AF was determined by MALDI-TOF to be 2% G0-F.

The carboxymethylated sensor chip surface matrix was activated byinjecting 20 μl of a mixture of 0.025 M N-hydroxysuccinimide and 0.1 MN-ethyl-N′(dimethylaminopropyl) carbodimide at 5 μl/min. The Hu9.1RFIgG1 antibodies were prepared in 10 mM sodium acetate, pH 4.5, at 30μg/ml for immobilization. 15 μl of the Hu9.1RF IgG1 was injected usingthree 5 μl injections and 20 μl of the afucosylated. Hu9.1RF IgG1 wasinjected using sequential 5, 10, and 5 μl injections. After coupling,unoccupied sites on the chip were blocked by injecting 20 μl of 1Methanolamine, pH 8.5. PBS containing 0.05% polysorbate 20 was used asthe running buffer. For kinetic measurements, two-fold serial dilutionsof the BR3 ECD (1.95-1000 nM) in running buffer were injected over theflow cells for 2 minutes at a flow rate of 30 μl/min and the bound BR3ECD was allowed to dissociate for 20 minutes. The binding surface wasthen regenerated with two injections of 30 μl 10 mM HCl (pH 2.0). Flowcell one, which was activated but did not have Hu9.1RF IgG1 immobilized,was used as a reference cell. There was no significant non-specificbinding of BR3 ECD to flow cell one. Data were analyzed using the 1:1Langmuir binding model and the association and dissociation rateconstants were fitted simultaneously with the BIAevaluation software.

Binding kinetics of Hu9.1RF IgG1 and the afucosylated Hu9.1RF IgG1 toBR3 ECD were measured and the apparent kinetic parameters of the Hu9.1RFIgG1 antibodies binding to immobilized BR3 ECD are shown in Tables 9Aand 9B.

TABLE 9A Binding of anti-BR3 antibodies to BR3 ECD using 1 bivalentanalyte model for data analysis. Antibody in solution Ka (105/Ms) Kd(10-4/s) KD (nM) Rmax (RU) Hu9.1RF IgG1 6.58 3.39 0.52 520 Afucosylated5.32 1.49 0.28 542 Hu9.1RF IgG1 37 RU of BR3 ECD was immobilized.Samples were analyzed at 1.56-200 nM in 2-fold serial dilution, unlessspecified otherwise.

TABLE 9B Binding of anti-BR3 antibodies to BR3 ECD using 1:1 Langmuirmodel for data analysis. Antibody in solution Ka (106/Ms) Kd (10-5/s) KD(nM) Rmax (RU) Hu9.1RF IgG1 1.99 5.21 0.03 307 Afucosylated 1.49 5.410.04 312 Hu9.1RF IgG1

The apparent binding affinities obtained using the 1 bivalent analytemodel for data analysis are more reliable since the antibodies arebivalent. The apparent binding affinities obtained using the 1:1Langmuir binding model for data analysis are higher likely due to theavidity effects. The apparent kinetic parameters for BR3 ECD binding toimmobilized Hu9.1RF IgG1 antibodies are shown in Table 10.

TABLE 10 Binding of Br3 ECD WT to anti-BR3-1 antibody using 1:1 Langmuirmodel for data analysis. Amount Antibody on immobilized Ka Kd KD Rmaxchip (RU) (105/Ms) (10-4/s) (nM) (RU) Hu9.1RF IgG1 2015 9.95 2.53 0.25155 Afucosylated 1962 8.52 1.99 0.23 150 Hu9.1RF IgG1 Samples wereanalyzed at 1.95-1000 nM in 2-fold serial dilution.

In summary, Hu9-1RF IgG1 and afucosylated Hu9.1RF IgG1 gave similarapparent affinities by BIAcore.

Example 16 Binding of Afucosylated Hu9.1RF IgG1 Antibody to Fcγ Receptor

Binding of control and test materials to the human Fcγ receptors wasassessed using modified versions of procedures originally described byShields et al. (J Biol Chem 276:6591-604 (2001)). For test materials,varying levels of G0-F content from 2-20% were prepared by mixing 37%G0-F Hu9.1RF IgG1 (generated via RNAi-fucosyltransferase knockdown) with2% G0-F GLP-Hu9.1RF IgG1. The “37% G0-F is really “52% afucosylated” byadding G1-F content (see FIG. 7). The 37% G0-F starting materialincluded G1-F carbohydrates. The starting material was calculated to be52% afucosylated if both G1-F and G0-F carbohydrates are included in thecalculation. Monomeric IgG is capable of binding to the high-affinityreceptor FcγRIA (CD64); however, the low-affinity receptors FcγRIIA(CD32A), FcγRIIB (CD32B), and FcγRIIIA (CD16) require multimeric IgG forbinding. Therefore, for the low-affinity receptor binding assays,multimers of the control or test materials were formed by premixing theantibody with F(ab′)₂ fragment goat anti-human κ chain (Cappel, ICNPharmaceuticals, Inc.; Aurora, Ohio) at a 1:2 (w/w) ratio in assaybuffer (0.5% bovine serum albumin [BSA], 0.05% polysorbate 20 inphosphate-buffered saline [PBS]) at pH 7.4. FcγRs were expressed asrecombinant fusion proteins of the extracellular domain of the receptoralpha chains with gly/his6/glutathione-s-transferase (GST). Assay platescoated with anti-GST and blocked with BSA were used to capture eachFcγR. Plates were washed with wash buffer (0.05% polysorbate 20 in PBS,pH 7.4) after each incubation. Serial dilutions of Hu9.1RF IgG1 orafucosylated Hu9.1RF IgG1 presented as monomers for FcγRIA and asmultimers for the low-affinity FcγR, were added to the plates induplicate and incubated for 2 hours. Bound antibody was detected withthe horseradish peroxidase (HRP)-conjugated F(ab′)2 fragment of a goatanti-human IgG F(ab′)₂ (Jackson ImmunoResearch; West Grove, Pa.). Plateswere developed with tetramethylbenzidine (TMB) (KPL, Inc.; Gaithersburg,Md.) as the substrate. Absorbance was measured at a wavelength of 450 nmwith a reference of 650 nm. Mean absorbance values from duplicate wellswere plotted as a function of antibody concentration. Data were fit to afour-parameter equation to determine the concentration that yields 50%of maximum binding to each FcγR (EC₅₀) for each antibody.

As shown in Table 11 and 12 below, no significant difference in bindingto FcγR1A, FcγRIIA and FcγRIIB was observed for the Hu9.1RF IgG1antibodies with variable levels of G0-F. The 100% AF Hu9.1RF IgG1 showedsignificantly higher binding affinities for FcγRIIIA-F and FcγRIIIA-V.

TABLE 11 Determined G0-F levels. Sample % G0-F (Expected Level of G0-F)(Determined level) Hu9.1RF IgG1, 2% 2 Hu9.1RF IgG1, 4% 3 Hu9.1RF IgG1,6% 6 Hu9.1RF IgG1, 10% 8 Hu9.1RF IgG1, 20% 23 HV3-46S-Bulk 4

TABLE 12 Average EC₅₀ for Hu9.1RF IgG1 with variable levels of G0-F.Average_(EC50) ng/mL μg/ml μg/ml μg/ml μg/ml n FcγR-IA FcγR-IIA FcγR-IIBFcγR-IIIA-F FcγR-IIIA-V Hu9.1RF IgG1 2% G0-F 3 3.92 0.30 0.96 2.20 0.34Hu9.1RF IgG1 4% G0-F 3 3.73 0.26 0.95 1.07 0.21 Hu9.1RF IgG1 6% G0-F 34.12 0.23 0.91 0.70 0.15 Hu9.1RF IgG1 10% G0-F 3 3.91 0.24 0.88 0.390.10 Hu9.1RF IgG1 20% G0-F 3 3.74 0.17 0.82 0.16 0.06 HV3-46S 1 1.580.30 1.02 1.52 0.28

Example 17 Binding of Afucosylated Hu9.1RF IgG1 Antibody to FcRn

As described above, Hu9.1RF IgG1 is a humanized monoclonal IgG1 antibodydirected against BR3 and having the VH and VL sequences of SEQ ID NOs:35 and 21, respectively (see Table 2). Like other IgG, antibodies, theFc portion of Hu9.1RF IgG1 binds to Fcγ receptors on the surfaces ofimmune effector cells. This binding initiates cellulariesponses such asantibody-dependent cellular cytotoxicity (ADCC) (Gessner et al., AnnHematol. 76:231-48 (1998)). Binding of Hu9.1RF IgG1 to the BR3 receptorblocks BAFF-dependent B-cell proliferation, and also induces Fc-mediatedcell killing through ADCC, which results in B-cell depletion.

FcRn is the major histocompatibility complex class I-related neonatal Fcreceptor and is responsible for the long half-life of circulating IgG.The Fc region of IgG binds to FcRn at acidic pH (pH 6.0-6.5) andreleases from the receptor at neutral pH (pH 7.4) (Raghavan et al.,Biochemistry 34:14649-57 (1995)). This process protects IgG fromdegradation and recycles IgG back into circulation. The binding affinityof an IgG for FcRn at acidic pH has been found to correlate with thehalf-life of the IgG in serum (Ghetie and Ward, Ann. Rev. Immunol.18:739-66 (2000)). As described above for Example 16, we have generatedan afucosylated Hu9.1RF IgG1 (Hu9.1RF IgG1 AF) using a cell line lackingfucosyltransferase. The experiments described below investigated whetherafucosylated Hu9.1RF IgG1 altered binding affinity to FcRn compared withHu9.1RF IgG1 that has normal glycans.

Test material Hu9.1RF IgG1 AF and control antibody Hu9.1RF IgG1 wereprovided by Genentech, Inc. as clear, colorless liquid solutions. Beforetheir use in the study, all materials were stored in a refrigerator setto maintain a temperature of 2° C.-8° C.

Soluble human FcRn was produced in Chinese hamster ovary cells. Bindingof control and test materials to human FcRn was assessed as described byShields et al. J. Biol. Chem. 277.26733-26740 (2001)). Briefly, assayplates coated with NeutrAvidinä (Pierce Biotechnology; Rockford, Ill.)and blocked with BSA were used to capture biotinylated FcRn in assaybuffer at pH 7.4. Plates were washed following each incubation step withwash buffer at pH 7.4. Subsequent incubation and wash steps were carriedout at pH 6.0. Serial dilutions of Hu9.1RF IgG1 AF or Hu9.1RF IgG1control antibody were added to the plates in duplicate and incubated for2 hours. Bound antibody was detected with the HRP-conjugated F(ab′)₂fragment of a goat anti-human IgG F(ab′)₂. Plates were developed withTMB as the substrate. Absorbance was measured at a wavelength of 450 nmwith a reference of 620 nm.

To evaluate the dissociation of bound IgG from FcRn at pH 7.4, the assaywas carried out in a similar manner as previously described, except withthe addition of a dissociation step. After the sample-incubation step,the plates were washed with wash buffer at pH 6.0. Assay buffer at pH6.0 or pH 7.4 was added to the plates and incubated at room temperaturefor 45 minutes. The assay was then continued as previously described.

Hu9.1RF IgG1 was used as a control for the relative binding affinityanalysis for FcRn. The optical density at 450 nm at the midpointabsorbance (mid-OD) of the Hu9.1RF IgG1 titration curve was calculatedby first dividing the difference in absorbance at the highest and lowestconcentrations by two, and then adding the absorbance reading at thelowest concentration. The corresponding concentrations of control andsamples at this mid-OD were determined from the titration curves using afour-parameter regression curve-fitting program (XLfit, ID BusinessSolutions Ltd.; Guildford, Surrey, UK). The Hu9.1RF IgG1 AF bindingaffinity relative to Hu9.1RF IgG1 was calculated by dividing the mid-ODconcentration of Hu9.1RF IgG1 by that of Hu9.1RF IgG1 AF

The impact of lack of fucose in the glycans of Hu9.1RF IgG1 AF antibodywas characterized by using an ELISA to measure the binding affinity ofHu9.1RF IgG1 AF and Hu9.1RF IgG1 for human FcRn at pH 6.0.Representative binding curves of the two antibodies to human FcRn at pH6.0 are shown in FIG. 16. Relative to Hu9.1 RF IgG1 that has normalglycans, Hu9.1RF IgG1 AF showed a 1.4-fold higher binding affinity forhuman FcRn (see Table 13).

We have found that the presence of aggregates in antibody samples mayincrease the apparent binding affinity for FcRn in this ELISA. Thepercent aggregate for Hu9.1RF IgG1 AF was determined to be 1.9%, whichis higher than that for Hu9.1RF IgG1 (0.2%). The slightly increasedbinding affinity of HU9.1RF IgG1 AF for FcRn as compared with thewild-type molecule Hu9.1RF IgG1 is likely attributable to the presenceof aggregates. Therefore, the absence of fucose did not significantlyalter Hu9.1RF IgG1 AF binding affinity to FcRn.

TABLE 13 Summary of Relative Binding Affinities in FcRn Binding AssaysConcentration at Mid-OD Relative Affinity to Antibody ng/mL nM^(a)Hu9.1RF IgG1 Hu9.1RF IgG1 48.8 0.33 1.0 Hu9.1RF IgG1 AF 33.8 0.23 1.4

For IgG to recycle back into circulation, it must dissociate from FcRnat physiological pH. The dissociation of Hu9.1RF IgG1 AF and Hu9.1RFIgG1 from FcRn at pH 7.4 was determined. The result indicated that bothHu9.1RF IgG1 AF and Hu9.1RF IgG1 dissociated from FcRn in assay bufferat pH 7.4, whereas the antibodies remained bound at pH 6.0 (see FIG. 17)

Although afucosylated Hu9.1RF IgG1 showed a slightly higher bindingaffinity for human FcRn at pH 6.0 in the FcRn ELISA, taken into theconsideration of aggregate effect on the assay, lack of fucose did notsignificantly affect Hu9.1RF IgG1 AF binding to FcRn compared withHu9.1RF IgG1 that has normal fucose content. Both Hu9.1RF IgG1 AF andHu9.1RF IgG1 antibodies bound to FcRn showed good dissociation atpH-7.4.

Example 18 Complement Dependent Cytotoxicity Assay Using AfucosylatedHu9.1RF IgG1 Antibody

The complement dependent cytotoxicity (CDC) assay measures the degree ofantibody dependent complement lysis of target cells. Human serumcomplement protein C1q binds to the Fc domain of an antibody bound toits target cells and triggers the initiation of the complement cascade.This action eventually culminates in the formation of the complementprotein membrane attack complex resulting in target cell lysis. Theassay is performed in a 96 microwell plate format and in duplicate asfollows.

For the experiments described below, AF Hu9.1 RF IgG1 was generatedusing a cell line lacking fucosyltransferase and is 100% afucosylated.50 μl of serially diluted (1:3) AF Hu9.1RF IgG1 and controls starting at300 nM was incubated with 50 μl of B-cell lymphoma cells lines (50,000)BJAB, and WIL2-S (ATCC CRL-8885), along with 50 μl of a 1:4 dilution ofnormal human serum complement (Quidel, Santa Clara, Calif.). After a 2hour incubation at 37° C., 50 μl of Alamar Blue (BiosourceInternational, Camarillo, Calif.) was added and incubated for anadditional 18 hours at 37° C. The plates were briefly shaken for 15minutes and then read on a fluorescent plate reader (Ext. 535 nm, Emt590 nm) to determine the relative fluorescent units (RFU). The RFU valueobserved was plotted relative to concentration of mAb in KaleidaGraph(Synergy Software, Reading, Pa.). Curves are plotted using a 4-parameterfit.

The CDC activity of AF Hu9.1RF IgG1 is shown in FIGS. 18A and 18B.Negative control and positive control monoclonal antibody were tested onBJAB and WIL2s cells. No complement dependent cytotoxicity was observedat any concentration of AF Hu9.1RF IgG1 on BJAB or WIL2-s cells.

Example 19 Antibody Dependent Cellular Cytotoxicity Using afucosylatedHu9.1RF IgG1 Antibody

As described in Example 7, above, the antibody dependent cellularcytotoxicity assay (ADCC) measures the degree of antibody dependent, NKcell killing of target cells. NK cells express on their surface the TypeIII Fc gamma receptor (CD16), a low affinity receptor for theimmunoglobulin Fc domain of antibodies. Antibodies that are bound totarget cells can bind CD16 with high avidity enabling the recruitment ofeffector NK cells whereupon NK cell activation leads to lysis of targetcells. NK cells are isolated from 100 mL of normal human whole bloodusing negative selection following the manufacture's (RosetteSep,StemCell Technologies) recommended protocol. Assays were performed inround bottom 96 microwell plates. The assay and all dilutions were inF12/DMEM containing 1% fetal bovine serum. In triplicate, 50 μl of 1:4serially diluted amounts of anti-BR3 and control monoclonal antibodies,starting at 100 nm, was incubated with 50 μl of target cells. Targetcells were BJAB (10,000 per 50 μL), a B-lymphoma cell line. After a 30minute room temperature incubation with the serially diluted antibodies,50 μl of effector NK cells (50,000) were added and incubated for anadditional 4 hours at 37° C. The effector to target cell ratio was 5:1.The plate(s) were centrifuged at 1500 rpm for 10 minutes and 100 μl ofthe cell media was removed and assayed. The level of cell lysis wasdetermined by measuring the amount of lactate dehyrogenase (LDH kit,Roche Diagnostics) released from lysed cells. The percent lysis relativeto mAb concentration was determined and plotted in KaleidaGraph (SynergySoftware, Reading, Pa.) using a 4-parameter curve fit.

As described in Example 18, AF Hu9.1 RF IgG1 was generated using a cellline lacking fucosyltransferase and is 100% afucosylated. The ADCCactivity of AF Hu9.1RF IgG1 was compared with 2% afucosylated Hu9.1RFIgG1 using normal human NK cells from three donors (FIGS. 19A-19C). TheEC_(so) of AF Hu9.1RF IgG1 was greater than 6-fold improved in ADCCactivity than 2% afucosylated Hu9.1RF IgG1. In addition, AF Hu9.1RF IgG1demonstrated a greater than 3-fold increase in percent cytotoxicity over2% afucosylated Hu9.1RF IgG1. The ADCC activity of Hu9.1RF IgG1monoclonal antibodies differing in percent afucosylation was alsocompared using NK cells isolated from three normal donors (FIGS.20A-20C). The level of ADCC activity increased as the level ofafuscoylation increased. This was observed for all three donors.

Example 20 Apoptosis Assay

BR3⁺ BJAB lymphoma cells were used to measure the ability of theanti-BR3 monoclonal antibody AF Hu9.1RF IgG1 to induce apoptosis asmeasured through Annexin V staining and propidium iodide dye exclusion(Cat# V-13241 Molecular Probes, Seattle, Wash.). As described in Example18, AF Hu9.1 RF IgG1 was generated using a cell line lackingfucosyltransferase and is 100% afucosylated. The BJAB cells werecultured in RPMI 1640 media (Gibco, Rockville, Md.) containing 10% fetalcalf serum (Biosource International, Camarillo, Calif.) and 2 mML-glutamine (Gibco). Prior to being assayed, the cells were washed twicein fresh media and then adjusted to a cell concentration of 2×10⁶ permilliliter. 100 μl of cells were added to 96 well microassays plates(Becton Dickinson, Palo Alto, Calif.) which contained 100 μl of apredetermined amount of AF Hu9.1RF IgG1, negative control IgG andpositive control IgG, along with F(ab)′₂ goat anti-human Fc (Cat #109-006-098 Jackson ImmunoResearch Labs, West Grove, Pa.). The finalmonoclonal antibody concentrations were 100, 10, 1.0, and 0.1 nM and theF(ab)′2 goat anti-human Fc molecule was set at twice the concentrationof the AF Hu9.1RF IgG1 and control antibodies. Each point was performedin duplicate. After a 24-hour incubation period at 37° C., the cellswere washed twice with phosphate buffered saline and then stained withAnnexin V and propidium iodide according to the manufacture'srecommendations. The staining patterns of the BJAB cells were analyzedby flow cytometry using a FACSCalibur Flow Cytometer (Becton DickinsonSan Jose, Calif.), and collected for period of 10-20 seconds and by thenumber of events. The data was reduced using the Cellquest Pro software(Becton Dickinson). BJAB cells that were positive for (1) Annexin Vstaining, (2) both Annexin V and propiduim iodide double-staining, and(3) the number of unstained live cells were scored and plotted usingKaleidaGraph graphing software (Synergy Software, Reading, Pa.).

AF Hu9.1RF IgG1, at concentrations ranging from 0.1 to 100 nM and in thepresence of an anti-IgG crosslinker, did not increase the level AnnexinV staining above background when incubated with BR3 positive BJAB cellsfor 20 hours (FIGS. 21A-21C). In addition, under the same conditions,there was no observed increase in Annexin V and Propidium Iodide surfacestaining. These data indicated that crosslinked AF Hu9.1RF IgG1 (0.1 to100 nM) does not induce apoptosis in BR3 positive BJAB cells.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

All publications, patent applications, and patents mentioned in thisspecification, including U.S. Provisional Application No. 60/830,969,are herein incorporated by reference to the same extent as if eachindependent publication, patent, or patent application was specificallyand individually indicated to be incorporated by reference.

1-35. (canceled)
 36. An anti-BR3 antibody having an Fc region whereinsaid anti-BR3 antibody is afucosylated.
 37. An anti-BR3 antibodycomposition comprising an afucosylated anti-BR3 antibody of claim 36.38. The anti-BR3 antibody composition of claim 37, wherein saidcomposition comprises at least 2% afucosylated anti-BR3 antibodies. 39.The anti-BR3 antibody composition of claim 39, wherein said compositioncomprises at least 4% afucosylated-anti-BR3 antibodies.
 40. The anti-BR3antibody composition of claim 39, wherein said composition comprises atleast 10% afucosylated anti-BR3 antibodies.
 41. The anti-BR3 antibodycomposition of claim 40, wherein said composition comprises at least 19%afucosylated-anti-BR3 antibodies.
 42. The anti-BR3 antibody compositionof claim 41, wherein said composition comprises 100% afucosylatedanti-BR3 antibodies.
 43. The afucosylated anti-BR3 antibody of claim 36,further comprising a variant Fc sequence, wherein the Fc sequence has asubstitution at any one or any combination of positions selected fromthe group consisting of 268D, 326D, 333A/334A, 298A/333A, 298A/334A,239D/332E, 239D/298A/332E, 239D/268D/298A/332E,239D/268D/298A/326A/332A, 239D/268D/298A/326A/332E,239D/268D/283L/298A/332E, 239D/268D/283L/298A/326A/332E, 239D/330L/332E,272Y/254T/256E, T250Q/M428L, D265A, and N297A, wherein the D265Asubstitution is in the absence of N297A and the N297A substitution is inthe absence of D265A.
 44. The afucosylated anti-BR3 antibody of claim36, further comprising a variant Fc sequence, wherein the antibodycomprises an Fc region that has been altered to change the ADCC, CDCand/or pharmacokinetic property of the antibody compared to a wild typeIgG Fc sequence by substituting an amino acid at any one or anycombination of positions selected from the group consisting of: 238,239, 246, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269,270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295,296, 297, 298, 301, 303, 305, 307, 309, 312, 314, 315, 320, 322, 324,326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373,376, 378, 382, 388, 389, 398, 414, 416, 419, 428, 430, 434, 435, 437,438 and 439 of the Fc region.
 45. The afucosylated anti-BR3 antibody ofclaim 36, wherein the antibody has ADCC activity in the presence ofhuman effector cells or has increased ADCC in the presence of humaneffector cells compared to an anti-BR3 antibody comprising a humanwildtype IgG1 Fc.
 46. (canceled)
 47. The afucosylated anti-BR3 antibodyof claim 36, wherein the antibody can block BAFF (SEQ ID NO:143) frombinding to the extracellular domain of BR3 (SEQ ID NO:151). 48.(canceled)
 49. The afucosylated anti-BR3 antibody of claim 36, whereinthe antibody binds an FcγRIII.
 50. The afucosylated anti-BR3 antibody ofclaim 36, wherein the antibody binds the FcγRIII with better affinity,or mediates antibody-dependent cell-mediated cytotoxicity (ADCC) moreeffectively, than the glycoprotein with a mature core carbohydratestructure including fucose attached to the Fc region of theglycoprotein. 51-53. (canceled)
 54. The afucosylated anti-BR3 antibodyof claim 36, wherein the fucosyltransferase gene is the FUT8 gene. 55.The afucosylated anti-BR3 antibody of claim 36, wherein the antibody isessentially free of bisecting N-acetylglucosamine (GlcNAc) attached tothe mature core carbohydrate structure.
 56. The afucosylated anti-BR3antibody of claim 36, wherein the antibody has bisectingN-acetylglucosamine (GlcNAc) attached to the mature core carbohydratestructure.
 57. The afucosylated anti-BR3 antibody of claim 36, whereinthe antibody has one or more galactose residues attached to the maturecore carbohydrate structure.
 58. The afucosylated anti-BR3 antibody ofclaim 36, wherein the antibody is essentially free of one or moregalactose residues attached to the mature core carbohydrate structure.59. The afucosylated anti-BR3 antibody of claim 36, wherein the antibodyhas one or more sialic acid residues attached to the mature corecarbohydrate structure.
 60. The afucosylated anti-BR3 antibody of claim36, wherein the antibody is essentially free of one or more sialic acidresidues attached to the mature core carbohydrate structure.
 61. Apharmaceutical composition comprising the antibody composition of claim37. 62-63. (canceled)
 64. The anti-BR3 antibody of claim 36, wherein theanti-BR3 antibody is a humanized or human antibody that binds to a humanBR3 extracellular domain sequence and has an H1, H2 and H3 region withat least 70% homology to the H1, H2 and H3 region, respectively, of anyone of the antibodies of Table 2 and has an L1, L2 and L3 region with atleast 70% homology to the L1, L2 and L3 region, respectively, of any oneof the antibodies of Table
 2. 65. The anti-BR3 antibody of claim 36,wherein the anti-BR3 antibody is a human or humanized antibody thatbinds to a human BR3 extracellular domain sequence and has at least 70%homology to a VH domain of any one of the antibodies of Table
 2. 66. Theanti-BR3 antibody of claim 36, wherein the anti-BR3 antibody is ahumanized antibody that binds to a human BR3 extracellular domainsequence, the antibody comprising an H3 sequence of any one of SEQ IDNOs. 4-13, 15, 16-18, 20, 22, 24, 26, 28-73, 75-76, 78, 80-85, 87-96,98, 100, 102, 104, 106, 107, 109-110, 112, 116, 118, 120, 122, 124-127and 129-131 and further comprising the H1 and H2 sequences and the L1,L2, and L3 sequences from any one of the antibodies disclosed in Table2. 67-69. (canceled)
 70. The anti-BR3 antibody of claim 36, wherein theanti-BR3 antibody has been conjugated to a cytotoxic agent or achemotherapeutic agent.
 71. The anti-BR3 antibody of claim 70, whereinthe cytotoxic agent is a radioactive isotope or a toxin.
 72. Theanti-BR3 antibody of claim 36, wherein the antibody is a monoclonalantibody.
 73. The anti-BR3 antibody of claim 36, wherein the antibody isa humanized antibody.
 74. The anti-BR3 antibody of claim 36, wherein theantibody is derived from a human antibody sequence.
 75. The anti-BR3antibody of claim 36, wherein the antibody is a multi-specific antibody.76. An isolated nucleic acid molecule that encodes the antibody of claim36.
 77. An expression vector encoding the antibody of claim
 36. 78. Ahost cell comprising a nucleic acid molecule of claim
 76. 79. The hostcell of claim 78, that produces the antibody of claim
 36. 80. The hostcell of claim 78, which is a mammalian cell, a yeast cell, or a plantcell.
 81. A method of treating a BR3 positive cancer, comprisingadministering to a patient suffering from the cancer a therapeuticallyeffective amount of a composition of claim
 37. 82. A method of treatinga B cell neoplasm, comprising administering to a patient suffering fromthe neoplasm a therapeutically effective amount of a composition ofclaim
 37. 83. A method of treating an autoimmune disease, comprisingadministering to a patient suffering from the autoimmune disease atherapeutically effective amount of a composition of claim
 37. 84. Amethod of treating a cancer, comprising administering to a patientsuffering from the cancer a therapeutically effective amount of acomposition of claim
 37. 85. A method of depleting B cells from a mixedpopulation of cells comprising contacting the mixed population of cellswith a composition of claim
 37. 86. The method according to claim 83further comprising the step of administering a therapeutically effectiveamount of an anti-CD20 antibody sequentially or concurrently with theanti-BR3 antibody.
 87. The method according to claim 86, furthercomprising the step of contacting the mixed population with an anti-CD20antibody sequentially or simultaneously with the anti-BR3 antibody. 88.The method of claim 86, wherein the CD20 binding antibody is therituximab antibody.
 89. The method of claim 83, further comprising thesequential or concurrent administration of a therapeutically effectiveamount of at least one of the group consisting of: a BAFF antagonist, abiologic response modifier, a B cell depletion agent, a cytotoxic agent,a chemotherapeutic agent and an immunosuppressive agent.
 90. The methodof according to claim 89, wherein the BAFF antagonist is selected fromthe group consisting of BR3-Fc, TACI-Fc, BCMA-Fc, an anti-BAFFpeptibody, an anti-BAFF antibody and an anti-BR3 antibody. 91-92.(canceled)
 93. The method of claim 83, wherein the autoimmune disease isselected from the group consisting of rheumatoid arthritis, juvenilerheumatoid arthritis, systemic lupus erythematosus (SLE), Wegener'sdisease, inflammatory bowel disease, idiopathic thrombocytopenic purpura(ITP), thrombotic thrombocytopenic purpura (TTP), autoimmunethrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgMpolyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus,Reynaud's syndrome, Sjörgen's syndrome and glomerulonephritis.
 94. Themethod of claim 93, wherein the autoimmune disease is rheumatoidarthritis.
 95. The method of claim 89, wherein the immunosuppressiveagent is methotrexate.
 96. The composition of claim 37, wherein about20-100% of the anti-BR3 antibodies in the composition are afucosylated.